Pluripotent embryonic stem (ES) cells are a potential source of all types of cells for regenerative medicine. ES cells maintain pluripotency through a complex interplay of different signaling pathways and transcription factors, including leukemia inhibitory factor (LIF), Nanog, Sox2, and Oct3/4. Nanog, however, plays a key role in maintaining the pluripotency of mouse and human ES cells. Phosphoinositde 3-kinase (PI3K) signaling pathway which is activated in response to growth factors and cytokines also plays a critical role in promoting the survival and proliferation of ES cells. Our earlier studies revealed that retinol, the alcohol form of vitamin A, enhances the expression of Nanog and prevents differentiation of ES cells in long-term cultures. Normally vitamin A/retinol is associated with cell differentiation via its potent metabolite, retinoic acid. Thus far, no direct function has been ascribed to retinol itself. In this study, we demonstrate for the first time that retinol directly activates phosphoinositide three (PI3) kinase signaling pathway through IGF-1 receptor/insulin receptor substrate one (IRS-1) by engaging Akt/PKB-mTORC1 mammalian target of rapamycin-2 (mammalian target of rapamycin complex 2), indicating a growth factor-like function of vitamin A. Furthermore, ES cells do not express enzymes to metabolize retinol into retinoic acid and lack receptors for retinol transport into the cytoplasm, indicating that retinol signaling is independent of retinoic acid. This study presents a novel system to investigate how extracellular signals control the self renewal of ES cells which will be important for high-quality ES cells for regenerative medicine.