Cell-based bone tissue engineering strategies have been effectively applied toward the development of grafting templates for skeletal repair and regeneration, but remain limited by inadequate availability of a robust mineralizing cell source. Dermal fibroblasts have emerged as a particularly promising cell alternative because they are harvested from autologous donors through minimally invasive skin biopsy and display a high capacity for in vitro expansion. In the present study, we investigated retroviral gene delivery of the osteogenic transcription factor Runx2 as a mineralization induction strategy in primary dermal fibroblasts. We demonstrate that constitutive overexpression of Runx2 induced osteogenic gene expression and mineralized nodule deposition in fibroblasts cultured on 3-dimensional fibrous collagen disks in vitro. Fourier transform infrared analysis revealed that Runx2 expressing fibroblasts deposit a carbonate-containing, poorly crystalline hydroxyapatite, whereas control constructs did not contain biologically-equivalent mineral. Importantly, Runx2-transduced fibroblasts formed mineralized templates in vivo after implantation in a subcutaneous, heterotopic site, whereas minimal mineralization was evident in control constructs. Furthermore, immunohistochemical analysis indicated that Runx2-engineered cells co-localized with mineral deposits in vivo, suggesting that nodule formation primarily originated from transplanted donor cells. These results establish Runx2-genetic engineering as a strategy for the conversion of a non-osteogenic cellular phenotype into a mineralizing cell source for bone repair applications. Cellular therapies based on primary dermal fibroblasts would be particularly beneficial for patients with compromised ability to recruit endogenous osteoprogenitors to the site of injury as a result of extreme trauma, age, radiation treatment, or osteolytic disease.