Phosphoinositide 3-kinase (PI3K) is activated by transmembrane tyrosine kinases such as vascular endothelial growth factor (VEGF) receptors and Tie2 (tunica intima endothelial kinase 2), both of which are key regulators of vascular development. However, the in vivo role of PI3K during developmental vascularization remains to be defined. Here we demonstrate that mice deficient in the p110alpha catalytic subunit of PI3K display multiple vascular defects, including dilated vessels in the head, reduced branching morphogenesis in the endocardium, lack of hierarchical order of large and small branches in the yolk sac, and impaired development of anterior cardinal veins. These vascular defects are strikingly similar to those in mice defective in the Tie2 signaling pathway. Indeed, Tie2 protein levels were significantly lower in p110alpha-deficient mice. Furthermore, RNA interference of p110alpha in cultured endothelial cells significantly reduced Tie2 protein levels. These findings raise the possibility that PI3K may function as an upstream regulator of Tie2 expression during mouse development.