NRG-1β (neuregulin-1β) serves multiple functions during embryonic heart development by signalling through ErbB family receptor tyrosine kinases (ErbB2, ErbB3 and ErbB4). Previous studies reported that NRG-1β induces cardiomyogenesis of mESCs (mouse embryonic stem cells) at the later stages of differen-tiation through ErbB4 receptor activation. In the present study we systematically examined NRG-1β induction of cardiac myocytes in mESCs and identified a novel time window, the first 48 h, for NRG-1β-based cardiomyogenesis. At this time point ErbB3, but not ErbB4, is expressed. In contrast with the later differentiation of mESCs in which NRG-1β induces cardiomyogenesis via the ErbB4 receptor, we found that knocking down ErbB3 or ErbB2 with siRNA during the early differentiation inhibited NRG-1β-induced cardiomyogenesis in mESCs. Microarray analysis of RNA expression at this early time point indicated that NRG-1β treatment in mESCs resulted in gene expression changes important to differentiation including up-regulation of components of PI3K (phosphoinositide 3-kinase), a known mediator of the NRG-1β/ErbB signalling pathway, as well as activation of CREB (cAMP-response-element-binding protein). Further study demonstrated that the NRG-1β-induced phosphorylation of CREB was required for cardiomyogenesis of mESCs. In summary, we report a previously unrecognized role for NRG-1β/ErbB3/CREB signalling at the pre-mesoderm stage for stem cell cardiac differentiation.