Vaclav Vytlacil was born in New York City on November 1, 1892. In 1906 he studied at the Art Institute of Chicago, and in 1913, on a scholarship from Art Student League, he returned to New York City and studied under portrait painter John C. Johansen. Vytlacil's first teaching position was at the Minneapolis School of Art from 1916 to 1921, when he traveled to Paris to study the art of Cezanne. While in Europe Vytlacil visited relatives in Prague and decided to settle in Munich, where he enrolled in the Royal Academy of Art. At the Academy Vytlacil met two other Americans, Ernst Thurn and Worth Ryder. When Thurn left the Academy to study with abstractionist Hans Hofmann, Vytlacil followed shortly thereafter, and in 1924 the two organized the Hofmann summer school on Capri.

On August 18, 1927, Vytlacil married Elizabeth Foster in Florence. In 1928 they returned to the United States for one year, where Vytlacil lectured at the University of Berkeley and joined the Art Students League. Vytlacil returned to the United States permanently in 1935 and accepted a teaching position at the Florence Cane School in Rockefeller Center, New York City. The following year his only child, Anne Bozena, was born and Vytlacil helped found American Abstract Artists.

During his career, Vytlacil taught at the Art Students League, Queens College in New York, Black Mountain College in North Carolina, College of Arts and Crafts in California. In 1946, he rejoined the Arts Student League until his retirement in 1978. His first major Pompeiian painting was shown in 1951 at the Feigle Gallery in New York City and was later sold to the Pennsylvania Academy of Fine Arts. In 1975, the Montclair Art Museum in New Jersey held a retrospective exhibition. Vaclav Vytlacil died in 1984 at the age of 92.