VEGF and PI3kinase (PI3K) as well as protein kinase C (PKC)-regulated signaling in cardiac and vascular differentiation was investigated in ES cell-derived Flk-1⁺ cardiovascular progenitor cells. Flk-1⁺ were cells isolated from 4-day-old EBs and cultivated in the presence or absence of VEGF from day 4 until day 10. Furthermore these cells were exposed to PI3K and PKC inhibitors. A clear reduction of vascular tube formation and reduction of mRNA expression analysing typical endothelial (CD31 and VE-Cadherin) as well as early (Nkx2.5 and GATA4) and late (MLC2v, cTnT, a-MHC and ß-MHC) cardiac genes were found after exposure to the PI3K inhibitor wortmannin and LY294002, the p110a inhibitor compound 15e and the p110 d inhibitor IC-87114. These effects were not abolished by application of VEGF. In addition, the results showed that vasculogenesis but not cardiomyogenesis was decreased in Flk-1⁺ cells preincubated with antagonists of the PKC family BIM-1, GÖ 6976 (targeting PKCa/bII) and rottlerin (targeting PKCd). In contrast myr- PKCz peptide (targeting PKCz) did not influence cardiac and vascular differentiation in ES cells. In summary we demonstrate that PI3K catalytic subunits p110a and p110 d 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.