Integrins of the beta1 subfamily are highly expressed in the early mouse embryo and are essential for the formation of primitive germ layers from the inner cell mass (ICM). We investigated the mechanisms by which alphabeta1 integrins regulate ICM morphogenesis by using the embryonic-stem-cell-derived embryoid body (EB), a model for peri-implantation development. Ablation of integrin beta1 in EBs resulted in endoderm detachment and in maturation defects, which were manifested by the mislocalization of GATA4 in the cytoplasm and the markedly reduced synthesis of basement membrane (BM) components and the lineage marker disabled homolog 2. The mutant endoderm cells failed to spread on BM substrates, but could spread on vitronectin, which induced upregulation of alphavbeta3 integrin and integrin-dependent GATA4 nuclear translocation. Forced expression of integrin beta3 in the mutant EBs completely rescued endoderm morphogenesis, suggesting that integrin beta3 can substitute for integrin beta1 in the endoderm. Furthermore, the mitogen-activated protein kinases (MAPKs) ERK1 and ERK2 (ERK1/2) and p38 were activated in endoderm in an integrin-dependent fashion. Pharmacological inhibition of ERK1/2 or p38 MAPK blocked vitronectin-induced GATA4 nuclear translocation and endoderm maturation, whereas expression of a constitutively active ERK kinase (MEK1) or p38 MAPK in the mutant cells rescued endoderm maturation in integrin-beta1-null endoderm cells. Collectively, these results suggest that integrins are required for both the stable adhesion and maturation of visceral endoderm, the latter being mediated through the activation of ERK1/2 and p38 MAPK.