Tissue engineering has emerged as a promising strategy to generate bone-grafting substrates. These approaches, however, are limited by an insufficient supply of committed osteoprogenitor cells and dedifferentiation of osteogenic cells during in vitro culture. To address these limitations, we engineered bone marrow stromal cells to constitutively express the osteoblastic transcription factor Runx2/Cbfa1, using retroviral gene delivery. These Runx2-modified cells were integrated into three-dimensional polymeric scaffolds to create tissue-engineered constructs. Compared with control stromal cells, Runx2 overexpression significantly upregulated osteoblastic differentiation and mineralization in vitro and in vivo in an ectopic, nonosseous subcutaneous site. More importantly, in vitro construct development to create a mineralized template before implantation dramatically enhanced subsequent in vivo mineralized tissue formation, providing a novel templating tissue-engineering strategy to improve in vivo mineralization. Finally, Runx2 overexpression and in vitro construct development synergistically enhanced in vivo mineralization compared with in vitro construct development or genetic engineering alone. This work provides a novel integrated genetic and tissue-engineering strategy to create mineralized templates for generating robust bone-grafting material.