The signal transducer and activator of transcription 5 (STAT5) is an essential factor in the signal transduction pathways for a number of cytokines that regulate the growth and differentiation of mammalian cells. In this study, we investigated the STAT5 signaling pathway in mouse embryos, to elucidate the mechanism of cytokine signal transduction that regulates preimplantation development. The results of the RT-PCR analysis showed that both STAT5A and B were expressed throughout preimplantation development. Immunocytochemistry revealed that the STAT5A/B proteins were located in the nucleus from the early 1-cell stage to the blastocyst stage. STAT5 activation appeared to be regulated by Janus kinases (JAKs) and SRC family kinases (SFKs), since inhibitors of these kinases inhibited the localization of STAT5 proteins to the nucleus. The JAK inhibitor Ag490 reduced both the developmental rate of the embryos and the expression levels of the downstream genes of the JAK-STAT5 signaling pathway. These findings suggest that STAT5 proteins function in preimplantation development by mediating the signals from cytokines.