PUFAs and their metabolites may influence differentiation processes of stem cells. The aim of the current study was to determine the effects of LA as ω-6-PUFA and EPA as ω-3- PUFA on vasculogenesis and cardiomyogenesis of ES cells. LA and EPA increased vascular structure formation and protein expression of the endothelial-specific markers ... FLK-1, PECAM-1, and VE-Cadherin, whereas the saturated fatty acid PA was without effect. LA and EPA increased ROS and NO, activated eNOS and raised intracellular calcium. The calcium response was inhibited by the intracellular calcium chelator BAPTA-AM and SSO, which is an antagonist of CD36, the scavenger receptor for fatty acid uptake. Comparable effects were achieved with CD36-neutralizing antibodies. In a dose-dependent manner, LA and EPA enhanced cardiomyocyte differentiation and function. LA and EPA accelerated beating frequency of cardiac cells cluster (foci), increased the percentage of contracting EBs, the area of α-actinin-positive cardiac cells (beating area), and protein expression of the cardiac-specific markers α-actinin and MLC2a. Prevention of ROS generation by radical scavengers or the NADPH oxidase inhibitor VAS2870 blunted vasculogenesis and cardiomyogenesis of ES cells. Moreover, inhibition of eNOS by L-NAME abolished vasculogenesis of ES cells. PUFAs stimulated AMPK-α as well as PPAR-α. AMPK-α activation was abolished by calcium chelation as well as inhibition of ROS and NO generation. Moreover, PUFA induced vasculogenesis and cardiomyogenesis were blunted by the PPAR-α inhibitor GW6471. In conclusion, the data demonstrated that, LA as well as EPA, enhance cardiomyogenesis and vasculogenesis by mechanisms involving ROS, NO and changes in intracellular calcium, which are locked to the energy sensors AMPK-α and PPAR‐α.