Background: Apoptosis of vascular endothelial cells plays a central role in angiogenesis and atherosclerosis. This study investigates the molecular mechanisms of endothelial apoptosis induced by tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) following inhibition of phosphatidylinositol 3-kinase (PI3K). It examines downstream regulation and activation of the extrinsic and intrinsic pathways. Methods and Results: By flow cytometry, TRAIL receptors 2 and 3 were present to a greater extent than receptors 1 and 4. TRAIL reduced cell numbers in combination with the PI3K inhibitor LY 294002. TRAIL (100 ng/ml) with LY 294002 (20 µmol/l) activated the extrinsic pathway, causing progressive cleavage of caspase-8 and caspase-3. Activation of the intrinsic pathway proceeded by release of mitochondrial factors Smac/DIABLO and cytochrome c, and caspase-9 cleavage. LY 294002 reduced phosphorylated Akt (p-Akt), with early loss of the short form of cellular FLIP (c-FLIP<sub>S</sub>) and concurrent reduction of Bcl-2. Treatment with small interfering RNA against PI3K also reduced c-FLIP<sub>S</sub> and Bcl-2, and cotreatment with TRAIL triggered caspase-3 cleavage. Conclusions: This study details the molecular regulation of TRAIL-induced apoptosis in vascular endothelium. Inhibition of PI3K reduces p-Akt, with concurrent reductions in c-FLIP<sub>S</sub> and Bcl-2, and so renders endothelium sensitive to TRAIL-induced apoptosis through the extrinsic and intrinsic pathways.