Early lineage segregation in preimplantation embryos and maintenance of pluripotency in embryonic stem cells (ESCs) are both regulated by specific signaling pathways. Small molecules have been shown to modulate these signaling pathways. We examined the influence of several small molecules and growth factors on secondlineage segregation of the inner cell mass toward hypoblast and epiblast lineage during mouse embryonic preimplantation development. We found that the second-lineage segregation is influenced by activation or inhibition of the transforming growth factor (TGF)b pathway. Inhibition of the TGFb pathway from the twocell, four-cell, and morula stages onward up to the blastocyst stage significantly increased the epiblast cell proliferation. The epiblast formed in the embryos in which TGFb signaling was inhibited was fully functional as demonstrated by the potential of these epiblast cells to give rise to pluripotent ESCs. Conversely, activating the TGFb pathway reduced epiblast formation. Inhibition of the glycogen synthase kinase (GSK)3 pathway and activation of bone morphogenetic protein 4 signaling reduced the formation of both epiblast and hypoblast cells. Activation of the protein kinase A pathway and of the Janus kinase/signal transducer and activator of transcription 3 pathway did not influence the second-lineage segregation in mouse embryos. The simultaneous inhibition of three pathways-TGFb, GSK3b, and the fibroblast growth factor (FGF)/extracellular signalregulated kinases (Erk)-significantly enhanced the proliferation of epiblast cells than that caused by inhibition of either TGFb pathway alone or by combined inhibition of the GSK3b and FGF/Erk pathways only.