We hypothesized that PDGF-B/PDGFR-β-signaling is important in the cardiac contribution of epicardium-derived cells and cardiac neural crest, cell lineages crucial for heart development. We analyzed hearts of different embryonic stages of both Pdgf-b−/− and Pdgfr-β−/− mouse embryos for structural aberrations with an established causal relation to defective contribution of these cell lineages. Immunohistochemical staining for αSMA, periostin, ephrinB2, EphB4, VEGFR-2, Dll1, and NCAM was performed on wild-type and knockout embryos. We observed that knockout embryos showed perimembranous and muscular ventricular septal defects, maldevelopment of the atrioventricular cushions and valves, impaired coronary arteriogenesis, and hypoplasia of the myocardium and cardiac nerves. The abnormalities correspond with models in which epicardial development is impaired and with neuronal neural crest–related innervation deficits. This implies a role for PDGF-B/PDGFR-β-signaling specifically in the contribution of these cell lineages to cardiac development. Developmental Dynamics 237:494–503, 2008. © 2008 Wiley-Liss, Inc.