Ambrose was born in Lovington, Illinois, to Rosepha Trippe Ambrose and Stephen Hedges Ambrose. His father was a physician who served in the U.S. Navy during World War II.

Ambrose was the lone historian featured in the 1973-74 ITV television series, The World at War, which detailed the history of World War II.  He served as the historical consultant for the movie Saving Private Ryan. Tom Hanks, who starred in the movie, said he "pored over D-Day" and Band of Brothers in researching his role. Hanks also credited Ambrose's books with providing extensive detail, particularly regarding D-Day landings.  The HBO mini-series, Band of Brothers (2001), for which he was an executive producer, helped sustain the fresh interest in World War II that had been stimulated by the 50th anniversary of D-Day in 1994 and the 60th anniversary in 2004. Ambrose served as executive producer for Price for Peace, a documentary concerning the war in the Pacific theater during World War II, and for Moments of Truth, a TV documentary containing interviews with World War II veterans.  In addition, Ambrose served as a commentator for Lewis & Clark: The Journey of the Corps of Discovery, a documentary by Ken Burns. He provided commentary in twenty made-for TV documentaries, covering diverse topics, such as World War II, Lewis & Clark, and America's prominence in the 20th century. He also appeared as a guest on numerous TV programs or stations, including The Charlie Rose Show, C-Span programming, CNN programming, NBC's Today Show, CNBC's Hardball, and various programming on The History Channel and the National Geographic Channel. Ambrose's association with National Geographic stemmed, in part, from his designation as an Explorer-in-Residence by the Society.  In addition to his academic work and publishing, Ambrose operated a historical tour business, acting as a tour guide to European locales of World War II. Also, he served on the board of directors for American Rivers and was a member of the Lewis and Clark Bicentennial Council.

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