Nitric oxide (NO) is a key regulator of cardiomyogenesis of embryonic stem (ES) cells. However, signaling pathways involving the energy sensor AMP-activated protein kinase (AMPK) and/or mammalian target of rapamycin (mTOR) resulting in NO generation and stimulation of cardiomyogenesis are currently not known. Herein, the role of AMPK- versus mTOR-regulated signaling pathways and the impact of NO for cardiomyogenesis of mouse ES cells were investigated. Activation of AMPK by 5-amino-4-imidazolecarboxamide riboside (AICAr) or metformin as well as inactivation of AMPK by compound C (Comp C), siRNA ablation of AMPKα2, or exogenous ATP stimulated cardiomyogenesis of ES cells. Inhibition of AMPK by Comp C resulted in phosphorylation of mTOR and generation of NO. NO generation was likewise achieved when AMPK was either activated by AICAr or mTOR was inhibited by rapamycin, suggesting that NO generation occurred by two mutually active parallel signaling pathways, one being AMPK dependent and mTOR independent (AICAr pathway) and the other being AMPK independent and mTOR dependent (Comp C pathway). Consequently, cardiomyogenesis as well as NO generation was completely abrogated when ES cells were cultivated in the presence of rapamycin and Comp C, which inhibit both signaling pathways. The impact of NO for cardiomyogenesis of ES cells was corroborated in experiments showing that the effects of Comp C on cardiomyogenesis of ES cells were abolished by the NO synthase inhibitors NG-monomethyl-l-arginine and N (G)-nitro-l-arginine methyl ester. In summary, our data demonstrate that NO generation downstream of AMPK and mTOR is activated by distinct, interacting signaling pathways that initiate cardiomyogenesis of ES cells.