In August 2004, Chambers was cast as surgical intern Alex Karev in Shonda Rhimes's medical drama series "Grey's Anatomy", opposite Ellen Pompeo, Sandra Oh, Katherine Heigl, and T. R. Knight. In 2006, along with other cast members, he was awarded a Screen Actors Guild Award for Outstanding Performance by an Ensemble in a Drama Series. In 2009, he also guest-starred on the "Grey's Anatomy" spin-off, "Private Practice", for the "Grey's Anatomy"/"Private Practice" crossover event. On January 10, 2020, Chambers officially announced his departure from "Grey's Anatomy" and that his final episode had aired on November 14, 2019, with a farewell episode airing on March 5, 2020.