Chondrogenesis and mechanical stimulation of the cartilage template, are essential for bone formation via the endochondral ossification process in vivo. Recent studies have demonstrated that in vitro regeneration strategies that mimic these aspects separately, either chondrogenesis or mechanical stimulation, can promote mineralization to a certain extent both in vitro and in vivo. However, to date no study has sought to incorporate both the formation of the cartilage template and the application of mechanical stimulation simultaneously to induce osteogenesis. In this study we test the hypothesis that mimicking both the biochemical and mechanical extracellular environment arising during endochondral ossification can enhance the in vitro mineralization potential of human Mesenchymal Stem Cells (hMSCs). hMSC cellular aggregates were cultured for 21 days under the following culture conditions; (1) Growth Medium-Hydrostatic Pressure (HP), (2) Chondrogenic Priming -HP, (3) Growth Medium +HP, (4) Chondrogenic Priming +HP. Each group was then further cultured for another 21 days in the presence of osteogenic growth factors without HP. Biochemical (DNA, sGAG, hydroxyproline, ALP, and calcium), histological (Alcian Blue, Alizarin Red), and immunohistological (Col I, II, X, BSP-2) analyses were conducted to investigate chondrogenic and osteogenic differentiation at various time points (14, 21, 35 and 42 days). Our results showed the application of HP induced chondrogenesis similar to that of chondrogenic priming but interestingly there was a reduction in hypertrophy markers (Collagen Type X) by applying HP alone versus chondrogenic priming alone. Moreover, the results showed that both chondrogenic priming and HP in tandem during the priming period, followed by culture in osteogenic medium accelerated the osteogenic potential of hMSCs.