It has been demonstrated that adult human circulating endothelial progenitor cells (EPCs) can differentiate to a cardiomyogenic phenotype. Notch signaling promotes epithelial-to-mesenchymal transformation and plays a role in heart and vessel development. Here, we investigated the role of Notch activation for cardiac differentiation of EPCs in a coculture system with neonatal cardiomyocyte. After coculture, Notch activation was transiently detected in EPCs, as determined by immunhistochemical detection of NICD (the intracellular cleavage fragment of Notch-1) and expression of human Notch target genes. Inhibition of γ-secretase blocked Notch cleavage and NICD translocation. Furthermore, the expression of the cardiac marker protein α-sarcomeric actinin and troponin T was significantly suppressed by γ-secretase inhibition or addition of soluble recombinant Jagged-1, indicating that Notch activation facilitates cardiac marker gene expression. Because noncanonical Wnts have previously been shown to promote cardiac differentiation, we additionally determined the influence of Notch activation on the expression of Wnt5a and Wnt11. Wnt5a and Wnt11 expression in the human cells was induced by the coculture and was blocked by γ-secretase inhibition. Likewise, stimulation of Notch signaling by immobilized Jagged-1 promoted Wnt5a expression in EPCs. These data suggest that Notch is activated upon coculture of EPCs with neonatal rat cardiac myocytes. γ-Secretase–dependent Notch activation is required for cardiac gene expression in human cells and induces the expression of noncanonical Wnt proteins, which may act in a paracrine manner to further amplify cardiac differentiation.