The E2f3 locus encodes two Rb-binding gene products, E2F3a and E2F3b, which are differentially regulated during the cell cycle and are thought to be critical for cell cycle progression. We targeted the individual inactivation of E2f3a or E2f3b in mice and examined their contributions to cell proliferation and development. Chromatin immunoprecipitation and gene expression experiments using mouse embryo fibroblasts deficient in each isoform showed that E2F3a and E2F3b contribute to G(1)/S-specific gene expression and cell proliferation. Expression of E2f3a or E2f3b was sufficient to support E2F target gene expression and cell proliferation in the absence of other E2F activators, E2f1 and E2f2, suggesting that these isoforms have redundant functions. Consistent with this notion, E2f3a(-/-) and E2f3b(-/-) embryos developed normally, whereas embryos lacking both isoforms (E2f3(-/-)) died in utero. We also find that E2f3a and E2f3b have redundant and nonredundant roles in the context of Rb mutation. Analysis of double-knockout embryos suggests that the ectopic proliferation and apoptosis in Rb(-/-) embryos is mainly mediated by E2f3a in the placenta and nervous system and by both E2f3a and E2f3b in lens fiber cells. Together, we conclude that the contributions of E2F3a and E2F3b in cell proliferation and development are context dependent.