The energy needs of the heart are met by fatty acid and glucose metabolism which are tightly regulated by the energy sensor AMP-activated protein kinase (AMPK). Although during embryogenesis the contracting heart requires an increased energy demand, the role of AMPK in heart development is not well elaborated. Using the embryonic stem (ES) cell model of cardiac cell differentiation it is shown that activation of AMPK by 5-amino-4-imidazolecarboxamide riboside (AICAr) as well as inactivation of AMPK by compound C (Comp C) stimulated cardiomyogenesis of ES cells and upregulated cardiac genes. Inhibition of AMPK by Comp C resulted in phosphorylation of mammalian target of rapamycin (mTOR) and generation of nitric oxide (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 signalling pathways, one being AMPK-dependent and mTOR-independent (AICAr pathway), the other being AMPK-independent and mTOR-dependent (Comp C pathway). Consequently, cardiomyogenesis as well as NO generation were completely abrogated when ES cells were cultivated in the presence of rapamycin and Comp C which should inhibit both signalling 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 nitric oxide synthase (NOS) inhibitor L-NMMA. In summary our data demonstrate that NO generation downstream of AMPK and mTOR is activated by distinct, interacting signalling pathways that initiate cardiomyogenesis of ES cells.