Vascular development and maturation are dependent on the interactions of endothelial cell integrins with surrounding extracellular matrix. Previous investigations of the primacy of certain integrins in vascular development have not addressed whether this could also be a secondary effect due to poor embryonic nutrition. Here, we show that the α₅ integrin subunit and fibronectin have critical roles in blood vessel development in mouse embryos and in embryoid bodies (EBs) differentiated from embryonic stem cells (a situation in which there is no nutritional deficit caused by the mutations). In contrast, vascular development in vivo and in vitro is not strongly dependent on α_v or β₃ integrin subunits. In mouse embryos lacking α₅ integrin, greatly distended blood vessels are seen in the vitelline yolk sac and in the embryo itself. Additionally, overall blood vessel pattern complexity is reduced in α₅-null tissues. This defective vascular phenotype is correlated with a decrease in the ligand for α₅ integrin, fibronectin (FN), in the endothelial basement membranes. A striking and significant reduction in early capillary plexus formation and maturation was apparent in EBs formed from embryonic stem cells lacking α₅ integrin or FN compared with wild-type EBs or EBs lacking α_v or β₃ integrin subunits. Vessel phenotype could be partially restored to FN-null EBs by the addition of whole FN to the culture system. These findings confirm a clear role for α₅ and FN in early blood vessel development not dependent on embryo nutrition or α_v or β₃ integrin subunits. Thus, successful early vasculogenesis and angiogenesis require α₅-FN interactions.