The impact of reactive oxygen species (ROS) and Phosphoinositide 3 kinase (PI3K) in vascular differentiating of embryonic stem (ES) cells is largely unknown. Here we show that silencing PI3K catalytic subunit p110α and NOX1 using short hairpin RNA (shRNA) or inhibition of Rac1 significantly abolished the superoxide (O2-) production stimulated by vascular endothelial growth factor (VEGF) in mouse ES cells and in ES cell-derived Flk-1+ vascular progenitor cells. Moreover, silencing p110α or inhibition the Rac1 arrested vascular development at initial stages of vasculogenesis in embryoid bodies even under VEGF treatment. In sorted Flk-1 positive ES cells tube-like structure formation on matrigel and cell migration in the scratch migration assay were totally impaired in absence of p110α, whereas silencing NOX1 in ES cells caused a significant reduction in the PECAM-1 positive area, branching points, cell migration and tube length upon VEGF treatment. However, vascular differentiation markers were still produced in shRNA VEGF treated NOX1 knock down ES cells. The efficiency of silencing the p110α catalytic subunit of PI3K and NOX1 to inhibit angiogenesis/vasculogenesis were investigated for their capacity to inhibit tumor-induced angiogenesis in confrontation cultures consisting of embryoid bodies and multicellular DU-145 prostate tumor spheroids. Interestingly, we found that silencing p110α can strongly inhibited the vascularization of multicellular tumor spheroids in confrontation cultures. These findings provide direct evidence that the activity of p110α in endothelial cells is essential in vasculogenesis/angiogenesis and suggest that p110α and their downstream signalling cascade may represent promising therapeutic targets for the treatment of numerous human diseases that involve aberrant neovascularization.