Bone marrow stromal cells represent a promising cell source for cell-based therapeutic and bone tissue-engineering applications, but are restricted by a low frequency in healthy marrow, an age-related decrease in osteogenic capacity, and a propensity for dedifferentiation during in vitro expansion. To address these limitations, retroviral gene delivery was used to examine the effects of sustained and elevated expression of the Runx2 osteoblastic transcription factor on osteoblastic gene and protein expression and mineralization in primary rat bone marrow stromal cells. Runx2 overexpression upregulated several osteoblast-specific genes, including collagen type I and osteocalcin, and enhanced alkaline phosphatase activity and biological mineral deposition. Forced Runx2 expression in combination with dexamethasone increased matrix mineralization compared with exogenous Runx2 expression or dexamethasone treatment alone, whereas dexamethasone-free control cultures displayed minimal mineralization. These additive effects suggest complementary interactions between Runx2 and dexamethasone-responsive regulatory factors. Finally, Runx2 overexpression in stromal cell cultures undergoing considerable in vitro expansion resulted in higher matrix mineralization capacity compared with controls, which completely lost the ability to produce mineralized matrix even in the presence of dexamethasone. These findings provide a novel strategy for cell-based therapeutic applications requiring significant numbers of osteogenic cells to synthesize mineralized constructs for the treatment of large bone defects.