This study investigated the differential effects of ramped and steady applications of cyclic hydrostatic pressure (CHP) on chondrogenic differentiation of bone marrow-derived human mesenchymal stem cells (hMSCs) in 3-dimensional culture in the absence of transforming growth factor-beta (TGF-beta). A custom hydrostatic pressure system was designed and manufactured. hMSCs were seeded in agarose and exposed to steady (7.5 MPa) or ramped (1 MPa to 7.5 MPa over a 14-day period) CHP for 4 h/d at f = 1 Hz for 14 days. Real-time reverse transcriptase polymerase chain reaction analysis was performed on days 0, 4, 9, and 14 to determine changes in messenger ribonucleic acid (mRNA) expression levels of Sox9, aggrecan, collagen I, and collagen II. Collagen II and aggrecan mRNA expression remained unchanged. Collagen I increased at day 4 in CHP specimens before decreasing to levels at or below same-day unloaded controls at days 9 and 14. On average, ramped and steady regimens of CHP increased Sox9, with the largest upregulation occurring at day 4 in response to steady pressure. These findings indicate that hydrostatic pressure may induce chondrogenesis in hMSC-seeded agarose constructs without TGF-beta, and that hMSCs are capable of withstanding high initial pressures that may initiate chondrogenesis faster than lower pressures.