He entered a federal courtroom shackled at the ankles and wearing a beige jail uniform over a long sleeve grey T-shirt.
He was not allowed to wear civilian clothing as allowed in state court. It appeared he has had a recent haircut.
With several rows of supporters looking on, Mangione stood and said "not guilty" when Judge Margaret Garnett asked how he pleaded to the four-count federal indictment charging him in the death of UnitedHealthcare CEO Brian Thompson.
Federal prosecutor Dominic Gentile said the government does not anticipate adding charges against Mangione.
Most of the evidence involves that which has already been collected by the Manhattan district attorney's office, which has charged Mangione with murder. He has pleaded not guilty.
Late Thursday night, federal prosecutors filed a required notice of their intent to seek the death penalty.
That came weeks after U.S. Attorney General Pam Bondi announced that she would be directing federal prosecutors to seek the death penalty for what she called "an act of political violence" and a "premeditated, cold-blooded assassination that shocked America."
Defense attorney Karen Friedman Agnifilo said she would seek to try only the federal case and eliminate the state case after federal prosecutors indicated they would seek the death penalty.
She said the defense would seek to preclude the government from pursuing the death penalty. The judge gave her a June 27 deadline to file her argument.
Mangione is next due in federal court December 5, at which time the judge said she would set a trial date sometime in 2026.
At the end of Friday's hearing, Agnifilo said prosecutors at the Manhattan district attorney's office told her they had inadvertently been "eavesdropping" on one of Mangione's calls with his lawyers.
Agnifilo said they were given a recording of the jailhouse call by federal prosecutors. Gentile said this was the first he was hearing of it.
The judge demanded a letter by next Friday with an explanation.
Mangione, an Ivy League graduate from a prominent Maryland real estate family, faces separate federal and state murder charges after authorities say he gunned down Thompson, 50, outside a Manhattan hotel on Dec. 4 as the executive arrived for UnitedHealthcare's annual investor conference.
The state murder charges carry a maximum punishment of life in prison.
The Associated Press and ABC News contributed to this report