Hydrostatic pressure (HP) is a key component of thein vivojoint environment and has been shown to enhance chondrogenesis of stem cells. The objective of this study was to investigate the interaction between HP and TGF-β3 on both the initiation and maintenance of a chondrogenic phenotype for joint tissue derived stem cells. Pellets generated from porcine chondrocytes (CCs), synovial membrane derived stem cells (SDSCs) and infrapatellar fat pad derived stem cells (FPSCs) were subjected to 10 MPa of cyclic HP (4 h/day) and different concentrations of TGF-β3 (0, 1 and 10 ng/mL) for 14 days. CCs and stem cells were observed to respond differentially to both HP and TGF-β3 stimulation. HP in the absence of TGF-β3 did not induce robust chondrogenic differentiation of stem cells. At low concentrations of TGF-β3 (1 ng/mL), HP acted to enhance chondrogenesis of both SDSCs and FPSCs, as evident by a 3-fold increase inSox9expression and a significant increase in glycosaminoglycan accumulation. In contrast, HP had no effect on cartilage-specific matrix synthesis at higher concentrations of TGF-β3 (10 ng/mL). Critically, HP appears to play a key role in the maintenance of a chondrogenic phenotype, as evident by a down-regulation of the hypertrophic markerstype X collagenandIndian hedgehogin SDSCs irrespective of the cytokine concentration. In the context of stem cell based therapies for cartilage repair, this study demonstrates the importance of considering how joint specific environmental factors interact to regulate not only the initiation of chondrogenesis, but also the development of a stable hyaline-like repair tissue.