The formation of endocardial cushions in the atrioventricular (AV) canal of the rudimentary heart requires epithelial-to-mesenchymal cell transformation (EMT). This is a complex developmental process regulated by multiple extracellular signals and transduction pathways. A collagen gel assay, long used to examine endocardial cushion development in avian models, is now being employed to investigate genetically engineered mouse models with abnormal heart morphogenesis. In this study, we determine interspecies variations for avian and mouse cultured endocardial cushion explants. Considering these observed morphologic differences, we also define the temporal requirements for TGFβ2 and TGFβ3 during mouse endocardial cushion morphogenesis. TGFβ2 and TGFβ3 blocking antibodies inhibit endothelial cell activation and transformation, respectively, in avian explants. In contrast, neutralizing TGFβ2 inhibits cell transformation in the mouse, while TGFβ3 antibodies have no effect on activation or transformation events. This functional requirement for TGFβ2 is concomitant with expression of TGFβ2, but not TGFβ3, within mouse endocardial cushions at a time coincident with transformation. Thus, both TGFβ2 and TGFβ3 appear necessary for the full morphogenetic program of EMT in the chick, but only TGFβ2 is expressed and obligatory for mammalian endocardial cushion cell transformation.