Born in 1935, Vlasta Pospíšilová graduated from the Academy of Arts, Architecture and Design in Prague in 1956, majoring in puppetry and stage design. As a fresh graduate she joined the Jiří Trnka Studio, where the celebrated long animated film A Midsummer Night's Dream / Sen noci svatojanske (1959) was being made.

Later she worked on other films by Jiří Trnka, including the short film Archangel Gabriel and Madam Goose / Archandel Gabriel a pani Husa (1965). She also did cutout stop motion animation and object animation, especially in Jan Švankmajer's films Jabberwocky / Zvahlav aneb Saticky Slameneho Huberta (1971) and Dimensions of Dialogue / Moznosti dialogu (1983).

As a film director she debuted in 1979 with the short puppet fairy tale About Mary and the Wolf Castle / O Marysce a Vlcim hradku, which was followed by over 45 films.