SummaryVEGF-, phosphoinositide 3-kinase (PI3K)-and protein kinase C (PKC)-regulated signaling in cardiac and vascular differentiation was investigated in mouse ES cells and in ES cell-derived Flk-1 + cardiovascular progenitor cells. Inhibition of PI3K by wortmannin and LY294002, disruption of PI3K catalytic subunits p110a and p110 using short hairpin RNA (shRNA), or inhibition of p110a with compound 15e and of p110 with IC-87114 impaired cardiac and vascular differentiation. By contrast, TGX-221, an inhibitor of p110b, and shRNA knockdown of p110b were without significant effects. Antagonists of the PKC family, i.e. bisindolylmaleimide-1 (BIM-1), GÖ 6976 (targeting PKCa/bII) and rottlerin (targeting PKC) abolished vasculogenesis, but not cardiomyogenesis. Inhibition of Akt blunted cardiac as well as vascular differentiation. VEGF induced phosphorylation of PKCa/bII and PKC but not PKCz. This was abolished by PI3K inhibitors and the VEGFR-2 antagonist SU5614. Furthermore, phosphorylation of Akt and phosphoinositidedependent kinase-1 (PDK1) was blunted upon inhibition of PI3K, but not upon inhibition of PKC by BIM-1, suggesting that activation of Akt and PDK1 by VEGF required PI3K but not PKC. In summary, we demonstrate that PI3K catalytic subunits p110a and p110 are central to cardiovasculogenesis of ES cells. Akt downstream of PI3K is involved in both cardiomyogenesis and vasculogenesis, whereas PKC is involved only in vasculogenesis.