Bone tissue engineering strategies are currently being developed as alternative mechanisms to address the clinical demand for bioactive and biomechanical graft material. To date, these efforts have been largely restricted by inadequate supply of committed osteoprogenitor cells and loss of osteoblastic phenotype expression following in vitro culture and expansion. The objective of this thesis research was to address the cell sourcing limitations of tissue-engineered bone grafts through constitutive and sustained overexpression of the osteoblast-specific transcriptional activator Runx2/Cbfa1 in osteogenic marrow-derived stromal cells using retroviral gene delivery. Runx2 overexpression enhanced expression of multiple osteoblastic genes proteins and, more importantly, significantly up-regulated matrix mineralization in both monolayer culture and following cell seeding in 3-D polymeric scaffolds. To evaluate in vivo performance, Runx2-expressing cells were seeded into 3-D constructs and implanted both subcutaneously and in a critical size craniotomy bone defect model. Notably, in vitro pre-culture of Runx2-transduced cell-seeded constructs prior to implantation significantly enhanced their capacity to form mineralized tissue in the subcutaneous space and induce new bone formation in the critical size defect model compared to control cells. The described series of analyses provided a novel combination of tissue and genetic engineering techniques toward the development of a Runx2-modified stromal cell/polymeric scaffold composite tissue-engineered bone graft substitute.