Polypyrimidine tract-binding protein (PTB) is a widely expressed RNA-binding protein with multiple roles in RNA processing, including the splicing of alternative exons, mRNA stability, mRNA localization, and internal ribosome entry site-dependent translation. Although it has been reported that increased expression of PTB is correlated with cancer cell growth, the role of PTB in mammalian development is still unclear. Here, we report that a homozygous mutation in the mouse Ptb gene causes embryonic lethality shortly after implantation. We also established Ptb(-/-) embryonic stem (ES) cell lines and found that these mutant cells exhibited severe defects in cell proliferation without aberrant differentiation in vitro or in vivo. Furthermore, cell cycle analysis and a cell synchronization assay revealed that Ptb(-/-) ES cells have a prolonged G(2)/M phase. Thus, our data indicate that PTB is essential for early mouse development and ES cell proliferation.