While at Ridgewood High School , Stamatopoulos wrote for his school variety show and was astounded he could get laughs with the material he had written. While attending Columbia College Chicago he performed a duo act with comedian Andy Dick, in which Stamatopoulos played a ventriloquist and Dick played a dummy under the influence of sleeping pills.

Stamatopoulos had written a spec script for a Simpsons episode, just out of love for the show, and Andy convinced him to submit to The Ben Stiller Show, which got him hired. He met and worked with Bob Odenkirk. After Stiller's show was canceled, he moved to New York and joined the writing staff of Late Night with Conan O'Brien, also with Odenkirk. During the 90's he wrote for Late Show with David Letterman, The Dana Carvey Show and Mr. Show with Bob and David. In 2000 Dino and Robert Smigel pitched Comedy Central a spin-off of Smigel's TV Funhouse SNL cartoons which would be a half-hour sketch comedy show. TV Funhouse premiered on 2000 and was canceled after one season. He later joined the writing staff on Mad TV.

He also worked on Tom Goes to the Mayor with Mr. Show alumnus Bob Odenkirk. He created the stop motion animated series Moral Orel for Adult Swim which premiered on Christmas of 2005 and aired its final episode in December 2008. He also does the voice of Drinky Crow on The Drinky Crow Show.

Stamatopoulos's newest stop motion series Mary Shelley's Frankenhole, currently in its first season on Adult Swim, began airing on June 27, 2010.

Since 2009, Stamatopoulos has been appearing in a recurring role as "Star-Burns" on the NBC comedy series Community. He also works as an occasional consultant and writing contributor on the show.