WASHINGTON—Two central witnesses in the probe of President Trump’s actions regarding Ukraine were removed from their posts Friday, two days after Mr. Trump was acquitted in the Senate impeachment trial.

Late Friday, Gordon Sondland said Mr. Trump ordered him removed from his post as U.S. ambassador to the European Union, effective immediately. Mr. Sondland gave some of the most-high-profile testimony in the impeachment inquiry. He told House lawmakers that he had urged Ukraine to announce investigations that would benefit...