The bidirectional signaling and hemostatic functions of platelet αIIbβ3 are regulated by kindlin-3 through interactions with the β3 cytoplasmic tail. Little is known about kindlin regulation of the related “vitronectin receptor,” αVβ3. These relationships were investigated in endothelial cells, which express αVβ3 and kindlin-2 endogenously. “β3ΔRGT” knock-in mice lack the 3 C-terminal β3 tail residues, whereas in “β3/β1(EGK)” mice, RGT is replaced by the corresponding residues of β1. The wild-type β3 tail pulled down kindlin-2 and c-Src in vitro, whereas β3ΔRGT bound neither protein and β3/β1(EGK) bound kindlin-2, but not c-Src. β3ΔRGT endothelial cells, but not β3/β1(EGK) endothelial cells, exhibited migration and spreading defects on vitronectin and reduced sprouting in 3-dimensional fibrin. Short hairpin RNA silencing of kindlin-2, but not c-Src, blocked sprouting by β3 wild-type endothelial cells. Moreover, defective sprouting by β3ΔRGT endothelial cells could be rescued by conditional, forced interaction of αVβ3ΔRGT with kindlin-2. Stimulation of β3ΔRGT endothelial cells led to normal extracellular ligand binding to αVβ3, pin-pointing their defect to one of outside-in αVβ3 signaling. β3ΔRGT mice, but not β3/β1(EGK) mice, exhibited defects in both developmental and tumor angiogenesis, responses that require endothelial cell function. Thus, the β3/kindlin-2 interaction promotes outside-in αVβ3 signaling selectively, with biological consequences in vivo.