Preimplantation mouse embryos simultaneously express receptors for leptin and leukemia inhibitory factor (LIF), both of which trigger activation of STAT3 (Signal Transducer and Activator of Transcription) protein. To examine the joint effects of leptin and LIF on embryonic development, we studied preimplantation development and activation of STAT3 signaling of mouse embryos after exposure to leptin and/or LIF in vitro. Two-cell mouse embryos (Day 2) were cultured in the presence of leptin and/or LIF. Significantly fewer leptin-exposed than control embryos hatched by Day 5 and by Day 6 of development. In addition, cells of leptin-exposed Day 5 blastocysts showed a higher rate of DNA fragmentation, which is a sign of apoptosis. Leukemia inhibitory factor alone had no effect on the rates of embryonic development or DNA fragmentation. Simultaneous exposure of embryos to leptin and LIF increased the proportion of hatching embryos and decreased the rate of apoptosis compared to embryos exposed to leptin only. Leptin treatment was associated with an increased phospho-STAT3-specific immunofluorescence in the cell membrane of blastocysts, which was not observed in LIF-exposed embryos. In conclusion, LIF modifies the effect of leptin during preimplantation embryo development in mice, presumably by interfering with activation of STAT3 signaling.