Embryonic stem cells (ESCs) are pluripotent cells derived from the inner cell mass (ICM) that are able to self-renew or undergo differentiation depending on a complex interplay of extracellular signals and intracellular factors. However, the feedback regulation of differentiation-dependent ESC self-renewal is poorly understood. Retinoic acid (RA), a derivative of vitamin A, plays a critical role in ESC differentiation and embryogenesis. In the present study, we demonstrate that short-term treatment of murine (m) ESCs with RA during the early differentiation stage prevented spontaneous differentiation of mESCs. The RA-treated cells maintained self-renewal capacity and could differentiate into neuronal cells, cardiomyocytes, and visceral endoderm cells derived from three germ layers. The differentiation-inhibitory effect of RA was mimicked by conditioned medium from RA-treated ESCs and was accompanied with up-regulated expression of leukemia inhibitory factor (LIF), Wnt3a, Wnt5a, and Wnt6. Such RA-induced prevention of ESC differentiation was attenuated by a neutralizing antibody against LIF or by a specific Wnt antagonist Fz8-Fc and was totally reversed in the presence of both of them. Furthermore, knock-down of beta-catenin, a component of the Wnt signaling pathway, by small interfering RNA counteracted the effect of RA. In addition, RA treatment enhanced expression of endodermal markers GATA4 and AFP but inhibited expression of primitive ectodermal marker Fgf-5 and mesodermal marker Brachyury. These findings reveal a novel role of RA in ESC self-renewal and provide new insight into the regulatory mechanism of differentiation-dependent self-renewal of ESCs, in which Wnt proteins and LIF induced by RA have the synergistic action. The short-term treatment of ESCs with RA also offers a unique model system for study of the regulatory mechanism that controls self-renewal and specific germ-layer differentiation of ESCs.