Transcriptional regulatory cascades during epicardial and coronary vascular development from proepicardial progenitor cells remain to be defined. We have used immunohistochemistry of human embryonic tissues to demonstrate that the TBX5 transcription factor is expressed not only in the myocardium, but also throughout the embryonic epicardium and coronary vasculature. TBX5 is not expressed in other human fetal vascular beds. Furthermore, immunohistochemical analyses of human embryonic tissues reveals that unlike their epicardial counterparts, delaminating epicardial-derived cells do not express TBX5 as they migrate through the subepicardium before undergoing epithelial-mesenchymal transformation required for coronary vasculogenesis. In the chick, Tbx5 is expressed in the embryonic proepicardial organ (PEO), which is composed of the epicardial and coronary vascular progenitor cells. Retrovirus-mediated overexpression of human TBX5 inhibits cell incorporation of infected proepicardial cells into the nascent chick epicardium and coronary vasculature. TBX5 overexpression as well as antisense-mediated knockdown of chick Tbx5 produce a cell-autonomous defect in the PEO that prevents proepicardial cell migration. Thus, both increasing and decreasing Tbx5 dosage impairs development of the proepicardium. Culture of explanted PEOs demonstrates that untreated chick proepicardial cells downregulate Tbx5 expression during cell migration. Therefore, we propose that Tbx5 participates in regulation of proepicardial cell migration, a critical event in the establishment of the epicardium and coronary vasculature.