Clinical imperatives for new bone to replace or restore the function of traumatized or bone lost as a consequence of age or disease has led to the need for therapies or procedures to generate bone for skeletal applications. However, current in vitro methods for the differentiation of human bone marrow stromal cells (HBMSCs) do not, to date, produce homogeneous cell populations of the osteogenic or chondrogenic lineages. As epigenetic modifiers are known to influence differentiation, we investigated the effects of the DNA demethylating agent 5-aza-2′-deoxycytidine (5-aza-dC) or the histone deacetylase inhibitor trichostatin A (TSA) on osteogenic and chondrogenic differentiation. Monolayer cultures of HBMSCs were treated for 3 days with the 5-aza-dC or TSA, followed by culture in the absence of modifiers. Cells were subsequently grown in pellet culture to determine matrix production. 5-aza-dC stimulated osteogenic differentiation as evidenced by enhanced alkaline phosphatase activity, increased Runx-2 expression in monolayer, and increased osteoid formation in 3D cell pellets. In pellets cultured in chondrogenic media, TSA enhanced cartilage matrix formation and chondrogenic structure. These findings indicate the potential of epigenetic modifiers, as agents, possibly in combination with other factors, to enhance the ability of HBMSCs to form functional bone or cartilage with significant therapeutic implications therein.