The closed-door testimony, coming after a congressional subpoena when the White House directed her not to appear, represented a significant win for Democrats in the face of a year's worth of Trump administration witnesses who have refused to cooperate with their investigations into the President.
Her testimony -- lasting nearly 10 hours -- may not be the linchpin of their potential impeachment case, but it signals that the White House's claim that the investigation is illegitimate may not persuade witnesses to balk at appearing before Congress.
In a blistering statement to the committee, Yovanovitch said she had been dismissed last spring because of pressure from Trump and "a concerted campaign against me."
She questioned whether the associates of Trump's personal lawyer Rudy Giuliani -- who were pushing for her ouster -- were looking to benefit financially from her removal, and warned that the State Department, where she still works, was being "attacked and hollowed out from within."
Yovanovitch said she had been informed by Deputy Secretary of State John Sullivan that Trump had lost confidence in her, adding that the State Department had "been under pressure from the President to remove me since the Summer of 2018."
Yovanovitch said she believed she had been removed because of "unfounded and false claims by people with clearly questionable motives," a reference to the effort led by Giuliani and his associates to remove her.
Yovanovitch did not answer after the hearing when CNN asked if Trump should be impeached and she wouldn't say whether she's concerned about retaliation.
A source familiar with the testimony said Yovanovitch had provided lawmakers with more details about the campaign Giuliani and his associates were orchestrating to remove her as ambassador.
Yovanovitch told lawmakers that she had heard from officials within the Ukrainian government that Giuliani was going around trashing her and making stuff up about her, according to the source. At the time, she didn't have a clear picture of what he was up to, the source added, but she's learned more recently about his efforts as new details have emerged.
"There were a lot of corrupt actors in Ukraine who wanted to get rid of her because she was effectively advancing the State Department's anti-corruption policies," said Democratic Rep. Tom Malinowski of New Jersey. "And at the same time there was a shadow foreign policy being run through Rudy Giuliani, who also wanted to get rid of her because she was standing in the way of that shadow foreign policy, and these two groups came together and found each other to be natural allies."
Multiple sources said Yovanovitch was not familiar with details about the freezing of Ukraine aid and setting up a meeting between Trump and Ukrainian President Volodymr Zelensky because she had been removed before those efforts were underway.
Sources from both parties said the opening statement that was reported near the beginning of her appearance Friday was the most revelatory part of her testimony.
Friday's deposition is a key part of the Democrats' impeachment inquiry into the President and Ukraine, which has been fueled by a whistleblower complaint alleging Trump sought help from Ukraine to investigate his political rival and the White House tried to cover it up.
Rep. Sean Patrick Maloney, a New York Democrat on the Intelligence Committee, told CNN's Wolf Blitzer that Yovanovitch "gave a gripping and emotional account of presidential abuse of power."
"This is a good person who served for more than 30 years in the Foreign Service, who was thrown to the wolves by Mr. Giuliani, who was representing the financial interest of his now-indicted associates, and by President Trump, who was advancing his political interest trying to get an investigation started in Ukraine of the Bidens," he said.
Rep. Denny Heck, a Washington state Democrat, said Yovanovitch's testimony was "powerful" and "impactful."
Asked if her testimony added to his concerns about the President's conduct, Heck said: "Yes, but we started in a pretty bad place. The walls are closing in."
House Intelligence Chairman Adam Schiff, a California Democrat, wrote in a letter to colleagues Friday that the impeachment inquiry was moving "with a sense of urgency" and the committees "expect to announce additional testimony from relevant witnesses in the coming days."
US Ambassador to the European Union Gordon Sondland says he will appear under subpoena, and two other State Department officials are scheduled to testify as well.
Republicans leaving the deposition with Yovanovitch challenged the Democrats on the process of the impeachment inquiry, arguing that the interview transcripts should be released and the House minority should have subpoena power. They charged that the White House was blocking people from testifying because House Democrats weren't following a formal process.
"The transcribed interview last week or the deposition this week is something that the American people should be able to see or hear, and the real question is why the secrecy?" said Rep. Jim Jordan of Ohio, the top Republican on the House Oversight Committee.
"How about the Democrats provide the Republicans and the President the same exact rights that they would demand if everything was reversed? You are talking about an impeachment of the President of the United States, and everything is going to happen behind closed doors, offering no protection whatsoever, no transparency, no due process," said Republican Rep. Lee Zeldin of New York.
Obtaining Yovanovitch's testimony wasn't easy for Democrats, however.
The three committees leading the impeachment probe said in a statement that the State Department, directed by the White House, had told Yovanovitch not to testify. In response, the Democrats issued a subpoena.
"This duly authorized subpoena is mandatory, and the illegitimate order from the Trump Administration not to cooperate has no force," said Schiff, Foreign Affairs Chairman Eliot Engel of New York and Oversight Chairman Elijah Cummings of Maryland. "As is required of her, the Ambassador is now complying with the subpoena and answering questions from both Democratic and Republican members and staff."
The White House, in talking points distributed to Capitol Hill and obtained by CNN, suggests that Yovanovitch could "breach her obligations" as a State Department employee by testifying because she does not have a Trump administration lawyer present.
"Only State Department lawyers would be able to provide Yovanovitch with the correct counsel on what is classified or privileged and without that counsel there is serious danger that she could breach her obligations as a current employee not to reveal such information without authorization," the White House wrote.
In her statement, Yovanovitch said that after being asked to extend her tenure until 2020, she was told in April to get "on the next plane" back to Washington.
Deputy Secretary of State Sullivan "also said that I had done nothing wrong and that this was not like other situations where he had recalled ambassadors for cause," she added.
On Friday, Trump announced his intention to nominate Sullivan as the next US ambassador to Russia.
Yovanovitch's statement included a warning that the State Department was being "attacked and hollowed out from within," urging the State Department leadership and Congress to take action to stop it.
"The harm will come when private interests circumvent professional diplomats for their own gain, not the public good," she said.
"The harm will come when bad actors in countries beyond Ukraine see how easy it is to use fiction and innuendo to manipulate our system," she added. "In such circumstances, the only interests that will be served are those of our strategic adversaries, like Russia, that spread chaos and attack the institutions and norms that the US helped create and which we have benefited from for the last 75 years."
Democratic Del. Eleanor Holmes Norton of Washington, DC, who sits on the Oversight Committee, told reporters after the first hour of Yovanovitch's interview that she sounded credible.
"She's as apolitical as I've heard anyone," Norton said. "She has been the object of false statements, and she's clearing that up."
Democrats are also probing how the push for an investigation into the Bidens was tied to Ukrainian efforts to arrange a one-on-one meeting between Trump and Zelensky and the freezing of foreign aid to Ukraine.
"I agree with you 100%," Zelensky responded.
Yovanovitch, a career member of the Foreign Service who has served in ambassadorships under three presidents, was sworn in as ambassador to Ukraine in August 2016.
At the time of her removal from the post in May, the State Department said in a statement that Yovanovitch was "concluding her three-year diplomatic assignment in Kyiv in 2019 as planned." But Democrats at the time accused the White House of carrying out a "political hit job" -- and the latest revelations about Ukraine have only fueled questions about her removal.
Yovanovitch is the second official interviewed by the Intelligence, Foreign Affairs and Oversight committees as part of the Democrats' impeachment inquiry -- and the first still serving in the Trump administration.
But given the Trump administration's stance that the impeachment probe is illegitimate, it's still unclear how many others will appear.
This story has been updated.
CNN's Clare Foran and Ellie Kaufman contributed to this report.