OBJECTIVE: Fibronectin (FN) is widely expressed during embryonic development, most prominently around developing vasculature. Studies of mice bearing an FN-null mutation have demonstrated that FN plays a role in vascular development, but the functions of the RGD motif of FN in vascular development remain unknown. METHODS AND RESULTS: Here we report that mouse embryos in which the RGD motif of FN had been replaced with an inactive RGE motif (FN(RGE/RGE) embryos) died as a result of vascular rupture and extensive bleeding. FN(RGE/RGE) embryos displayed multiple defects of angiogenesis, including failure of embryonic and yolk sac vasculature remodeling, defective placental blood vessel invasion, and defective heart development, although initial vacuolization occurred. Detailed histological examination of the embryos revealed endothelial cell sheet detachment from the underlying mesenchyme, and delayed differentiation or recruitment of vascular smooth muscle cells (VSMCs) around the heart and dorsal aorta. CONCLUSIONS: These findings demonstrate that although FN is essential for both vasculogenesis and angiogenesis, the RGD motif plays specific roles in angiogenesis, including vascular invasion, remodeling, stabilization, and maturation.