Heart valve formation is initiated by an epithelial–mesenchymal cell transformation (EMT) of endothelial cells in the atrioventricular (AV) canal. Mesenchymal cells formed from cardiac EMTs are the initial cellular components of the cardiac cushions and progenitors of valvular and septal fibroblasts. It has been shown that transforming growth factor β (TGFβ) mediates EMT in the AV canal, and TGFβ1 and 2 isoforms are expressed in the mouse heart while TGFβ 2 and 3 are expressed in the avian heart. Depletion of TGFβ3 in avian or TGFβ2 in mouse leads to developmental defects of heart tissue. These observations raise questions as to whether multiple TGFβ isoforms participate in valve formation. In this study, we examined the localization and function of TGFβ2 and TGFβ3 in the chick heart during EMT. TGFβ2 was present in both endothelium and myocardium before and after EMT. TGFβ2 antibody inhibited endothelial cell–cell separation. In contrast, TGFβ3 was present only in the myocardium before EMT and was in the endothelium at the initiation of EMT. TGFβ3 antibodies inhibited mesenchymal cell formation and migration into the underlying matrix. Both TGFβ2 and 3 increased fibrillin 2 expression. However, only TGFβ2 treatment increased cell surface β-1,4-galactosyltransferase expression. These data suggest that TGFβ2 and TGFβ3 are sequentially and separately involved in the process of EMT. TGFβ2 mediates initial endothelial cell–cell separation while TGFβ3 is required for the cell morphological change that enables the migration of cells into the underlying ECM.