Multiple signaling pathways converge to maintain pluripotency of embryonic stem cells (ESC) and the inner cell mass (ICM) of the early mammalian blastocyst. Surprisingly little is known about the mechanisms that control cell fate in the pre-implantation embryo, however. Understanding the controlled segregation of two lineages, the primitive endoderm (PE) and epiblast (EPI) from the ICM, is critical in understanding lineage differentiation and maintenance of pluripotency of both ESC and the ICM. It is therefore important to study the molecular pathways that maintain and initiate differentiation of the ICM, PE and EPI. Because of its accessibility, the mouse blastocyst is a valuable model to probe lineage segregation in mammals with the goal of applying this knowledge to understanding the essential steps involved in cell commitment and differentiation of other tissue types. In the current investigation, we examined the effects of modulating the FGF and MAP kinase signaling pathways in shifting the fate of ICM cells to either EPI or PE. Embryos were harvested from mating of B6C3F1 mice and cultured for 3.5-4 days to obtain blastocysts. Laser dissection was employed to isolate ICM, which were plated onto irradiated human fibroblast feeders and grown with and without inhibitors (FGF receptor inhibitor PD173074, Mek inhibitor PD0325901). LIF was added to both media. After 6 days in vitro, ESC colonies were obtained from all ICM explants grown in the presence of the inhibitors (10/10), while no colonies formed in control media lacking inhibitors (0/6). These results suggest that inhibition Fgf receptor and MAP kinase signaling preferentially directs the differentiation of ICM cells into an epiblast-like phenotype. Current investigations are in progress to characterize the gene expression profile and stability of the phenotype in these ESC. This research suggests both methods to improve ESC derivation and to control lineage differentiation of the blastocyst, ESC and derivation of induced pluripotent stem cells (iPSC).