Insulin receptor (IR)-mediated signaling is involved in the regulation of pluripotent stem cells, however its direct effects on regulating the maintenance of pluripotency and lineage development are not fully understood. To this end, we generated IR knock-out (IRKO) mouse induced pluripotent stem cells (miPSCs) from E14.5 mouse embryonic fibroblasts (MEFs) of global IRKO mice using a cocktail of four reprogramming factors (Oct4, Sox2, Klf4 and cMyc). iPSCs were maintained in a 2-inhibitor (2i) feeder-free system. Interestingly, expression of pluripotency markers including Klf4, Lin28a, Tbx3 and cMyc were upregulated while abundance of Oct4 and Nanog were enhanced by 4-fold and 3-fold respectively in IRKO iPSCs. Analyses of signaling pathways demonstrated downregulation of phospho-STAT3, pmTor and pErk, and an increase in the total mTor and Erk proteins in IRKO iPSCs. Stimulation with leukemia inhibitory factor (LIF) showed a 3-fold decrease of phospho-ERK in IRKO iPSCs (p<0.vs. C, n=3 clones/group). On the contrary, Erk phosphorylation was increased during in-vitro spontaneous differentiation of iPSCs lacking IR. Lineage specific directed differentiation of the iPSCs revealed that cells lacking IR showed enhanced expression of neuronal lineage markers (Pax6, Tubb3, Asci and Oligo2) while exhibiting a decrease in adipocyte (Fas, Acc, Pparg, Fabp4, C/ebpa and Fsp27) and pancreatic beta cell markers (Ngn3, Isl1 and Sox9) (p<0.05, n=3 clones/group). Further molecular characterization by phosphoproteomics confirmed the novel IR-mediated regulation of the global pluripotency network and several key proteins involved in various aspects of growth and embryonic development ((p<0.05, n=3 clones/group). Thus, IRKO iPSCs provide an opportunity to explore the crosstalk of insulin receptor signaling with key signaling pathways required for the maintenance of pluripotency and lineage determination. Disclosure M.K. Gupta: None. D.F. De Jesus: None. S. Kahraman: None. F. Shamsi: None. J. Hu: None. Y. Tseng: Other Relationship; Self; Chugai Pharmaceutical Co., Ltd.. Research Support; Self; MedImmune. R. Kulkarni: None.