Embryonic stem cells (ESCs) form descendants of all 3 germ layers when differentiated as aggregates, termed embryoid bodies. sellectchem In vivo, differentiation of cells relies on signals and morphogen gradients that supply instructive and positional cues, but do such gradients exist in embryoid bodies? We report here the establishment of anteroposterior polarity as well as the formation of the primitive streak-like The insulin-like growth factor 1 (IGF-1) receptor region inside the embryoid body, dependent on neighborhood activation with the Wnt pathway. On this region, cells undergo an epithelial-to-mesenchymal transition and differentiate into mesendodermal progenitors. Exogenous Wnt3a protein posteriorizes the embryoid body, leading to predominantly mesendodermal differentiation. Conversely, inhibiting Wnt signaling promotes anterior character and outcomes in neurectodermal differentiation. The activation of Wnt signaling and primitive streak formation needs external signals but is self-reinforcing soon after initiation. Our findings display the Wnt pathway mediates the nearby execution of a gastrulation-like process within the embryoid entire body, which displays an sudden degree of self-organization.