Stamos started his acting career with a role on the television show General Hospital as Blackie Parrish. Later he joined Full House as Adam Cochran, but he then changed the first name to Jesse before filming the premiere. During the second season he then changed his character's last name to Katsopolis to highlight his Greek heritage, which Stamos is very proud of. From 2006 to 2009, Stamos had a starring role on the NBC medical drama ER as Dr. Tony Gates. In September 2010 Stamos began a multi-episode arc as Dr. Carl Howell on the second season of the Fox series Glee.

He has subsequently appeared in numerous made-for-television films, stage productions and commercials. Stamos also has an active music career, having occasionally toured as a drummer with The Beach Boys, and having appeared on Broadway as the Master of Ceremonies in Cabaret. He expanded his career to include the role of J. Pierrepont Finch in Broadway's production of How to Succeed in Business Without Really Trying! In September 2009, he began playing the role of Albert in the Broadway revival of Bye Bye Birdie. On July 10, 2012, Stamos began performances on Broadway in Gore Vidal's The Best Man as Senator Joseph Cantwell.

Since 2011, John Stamos has appeared in a series of commercials for Dannon's Greek yogurt brand Oikos; including his first Superbowl commercial which was aired during Superbowl 2012.

In 2015, Stamos starred in the new Fox comedy sitcom "Grandfathered." He also starred as Geroge in the 2016 film, "My Big Fat Greek Wedding 2."

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