Stokowski was trained at the Royal College of Music, London, and Queen's College, Oxford, and held positions as an organist before becoming conductor of the Cincinnati (Ohio) Symphony from 1909 to 1912. He gained an international reputation as musical director of the Philadelphia Orchestra from 1912 to 1936, and he frequently conducted concerts with them until 1941. He introduced youth concerts and performed contemporary works by Gustav Mahler, Edgard Varèse, and Igor Stravinsky and conducted the world premiere of Charles Ives's Symphony No. 4 (1909–16). He also experimented with the placement of various sections of the orchestra and occasionally changed the written orchestration of the music he performed.

Organizer of the All-American Youth Orchestra, he conducted the NBC Symphony from 1941 to 1944 with Arturo Toscanini (Toscanini, Arturo) and inaugurated low-priced concerts at the New York City Center (1944–45). He directed the New York Philharmonic from 1946 to 1950 and the Houston (Texas) Symphony from 1955 to 1962; in 1962 he formed the American Symphony Orchestra in New York City.

Stokowski made three films with the Philadelphia Orchestra, including Walt Disney's Fantasia (1940), and wrote a book, Music for All of Us (1943). He also made symphonic transcriptions of Johann Sebastian Bach's works.