Introduced as a surgical intern at the fictional Seattle Grace Hospital, O'Malley worked his way up to resident level, while his relationships with his colleagues Meredith Grey (Ellen Pompeo), Cristina Yang (Sandra Oh), Izzie Stevens (Katherine Heigl) and Alex Karev (Justin Chambers) formed a focal point of the series.

The actor said the reason for his departure was due to a "breakdown in communication" with executive producer Shonda Rhimes, his character's lack of screen time,[11] and his decision to come out as gay.

[12] Knight received generally positive reviews for his performance as O'Malley, and garnered a nomination for Outstanding Supporting Actor in a Drama Series at the 59th Primetime Emmy Awards.

Knight starred as Leo Frank in a production of the musical Parade, which opened October 4, 2009, at the Mark Taper Forum in Los Angeles.

After his role on the hit series ended, Knight married his husband Patrick B. Leahy, a ballet dancer and writer, in Hudson, New York, on October 7, 2013.