Cartilage tissue engineering is a promising treatment for damaged or diseased cartilage that requires thorough understanding of influential parameters involved in chondrogenic differentiation. This study examined how 4-h application of cyclic hydrostatic pressure (CHP) of 5 MPa at 0.5 Hz could modulate chondroinduction of human adipose-derived mesenchymal stem cells (hAMSCs) in vitro. Four groups were examined including a negative control group, a chemical group treated by growth factor for 10 days, a mechanical group exposed to 4-h loading on the 10th day of pellet culture without any chondrogenic stimulator, and finally a chemical-mechanical group subjected to both growth factor and loading. Application of cyclic hydrostatic pressure increased the expression of chondrogenic genes, including sox9 and aggrecan to higher levels than those of the chemical group. This study indicates that cyclic hydrostatic pressure initiates and enhances the chondrogenic differentiation of mesenchymal stem cells with or without growth factors in vitro and confirms the important role of hydrostatic pressure during chondrogenesis in vivo.