Tricho-rhino-phalangeal syndrome (TRPS) is an autosomal dominant skeletal disorder caused by mutations of TRPS1. Based on the similar expression patterns of Trps1 and Gdf5, we hypothesized a possible functional interaction between these two molecules. Using a chondrogenic cell line (ATDC5), we investigated the association of Gdf5-mediated signaling pathways with Trps1 and the phenotypic changes of ATDC5 cells due to over-expression or suppression of Trps1. Treatment of cells with Gdf5 enhanced Trps1 protein levels and phosphorylation of p38 mitogen-activated protein kinase (MAPK) in a dose-dependent manner. Nuclear translocation of Trps1 was also induced by Gdf5. These effects were blocked by a dominant negative form of activin-linked kinase 6 (dn-Alk6) and by SB203580, an inhibitor of the p38 MAPK pathway. Conversely, Gdf5 expression was suppressed by the over-expression of Trps1. Trps1-overexpressing ATDC5 (O/E) cells differentiated into chondrocytes more quickly than mock-infected control cells, whereas cells transfected with dn-Alk6 showed slower differentiation. On the other hand, O/E cells showed an increase of apoptosis along with the up-regulation of cleaved caspase 3 and down-regulation of Bcl-2, whereas dn-Alk6 cells showed suppression of apoptosis. In conclusion, Trps1 acts downstream of the Gdf5 signaling pathway and promotes the differentiation and apoptosis of ATDC5 cells.