Objectives: b1 integrins are important regulators of vascular differentiation and development since their endothelial-specific deletion results in embryonic lethality. Therefore, in the present study we have investigated the molecular mechanisms underlying the prominent vascular abnormalities in b1 integrin deficient murine embryonic stem (ES) cells. Methods: We quantified the amount of endothelial clusters and vessel-like structures in b1 integrin deficient, wild-type and rescue EBs. Vascular tube formation by magnet associated cell (MAC) sorted endothelial cells was determined in Matrigel. In addition, the amount of proliferating, apoptotic and migrating endothelial cells was analyzed by immunohistochemistry and changes in AKT and eNOS protein levels were measured by Western Blot. Results: We found that vessel development was strongly defective in embryoid bodies (EBs) devoid of b1 integrin, as only primitive and short sprouts developed from clusters of vascular precursors in b1 integrin (-/-) EBs whereas complex network formation of endothelial tubes was found in wild-type EBs (amount of vascular sprouts at day 5+7: 40.4±7.4, n=38 (wild-type) vs 7.7±1.1, n=32 (b1 integrin (-/-)). The vascular defect was due to deficient b1 integrin expression in endothelial cells, as endothelial-specific re-expression rescued the phenotype entirely. The mechanism responsible for defective vessel formation was reduced endothelial cell migration and elongation which was assessed in migration assays and time-lapse microscopy. Moreover, endothelial cell numbers were reduced because the apoptosis vs proliferation rate increased during differentiation. This effect was due to an elevation of peNOS and pAKT signaling molecules, respectively. Conclusion: We conclude that endothelial b1 integrins are critical determinants of vessel formation and this effect depends on changes in migration, proliferation and apoptosis levels.