An epithelial–mesenchymal cell transformation (EMT) occurs during the development of endocardial cushions in the atrioventricular (AV) canal of the heart. This is a complex developmental process regulated by multiple extracellular signals and signal transduction pathways. It was recently shown that the transcription factor Slug is expressed in the AV canal and is required for initial steps of EMT. Treatment of AV canal explants with either antisense oligodeoxynucleotides toward Slug or anti-TGFβ2 antibody inhibited initial steps of EMT. Others have identified roles for HGF and BMP during EMT in the heart. Both HGF and BMP are known to regulate Slug in other cell types. To determine whether TGFβ2 or other signaling factors regulate Slug expression during EMT in the heart, we cultured AV canal explants in the presence of anti-TGFβ2 antibody, anti-TGFβ3 antibody, pertussis toxin, retinoic acid, noggin, or anti-HGF antibody. Only treatment with anti-TGFβ2 antibody or retinoic acid inhibited Slug expression in AV canal explants. Consistent with these data, we found that retinoic acid disrupted initial steps of EMT, while antagonists of BMP and HGF signaling disrupted later steps of EMT. Transfection of AV canal explants with Slug rescued the inhibitory effect of anti-TGFβ2 antibody but not retinoic acid on EMT. Slug is thus an essential target of TGFβ2 signaling during EMT in the developing chicken heart.