Embryonic stem cells (ESCs) have the potential to be used as unlimited resources for tissue replacement therapy, thereby compensating for organ donor shortage. To reach this goal, the molecular principles governing early differentiation events in the developing embryo need to be addressed, understood and properly implemented in vitro. Studies carried out in several vertebrate models have established that Nodal/Activin A, BMP, WNT and FGF signaling pathways regulate early embryo development and that these pathways are similarly used during germ layer formation by cultured ESCs. However, differences have also been identified in the way these pathways function or interact in mouse vs. human ESCs, making it sometimes difficult to extrapolate findings from one system to the other. In this review, we discuss and compare the role of the relevant signaling pathways and their crosstalk during undifferentiated growth and during the endoderm differentiation of mouse and human ESCs.