The discovery that the definitive hematopoietic stem cells (HSCs) derive from specialized regions of the endothelium, known as the hemogenic endothelium (HE), shed a good deal of light on HSC embryonic developmental processes. This knowledge opened up new possibilities for the design of new strategies to obtain HSCs in vitro from pluripotent stem cells (PSCs). Previous advances in this field have shown that the Wnt/β-catenin signaling pathway plays a key role in PSC-derived HSC formation. In this work, we identified lithium, a GSK3 inhibitor, as an element capable of stabilizing β-catenin and inducing ESC differentiation in the mesoderm lineage and subsequently in the HE, highly consistent with the role of Wnt agonists on ESC differentiation. ESCs treated with 10 mM lithium express CD31+, Sca-1+, Nkx2-5+ and Runx1+ cells characteristic of HE cells. The ability of lithium-treated ESCs to further derive into HSCs was confirmed after defined maturation, resulting in rounded cell aggregates positive for fetal and mature HSCs markers, confirming the endothelial to hematopoietic transition. Our results represent a novel strategy for generating HSC in vitro as a multipotent source of stem cells for blood and muscle disease therapies.