Towards utilizing gene-targeted, repopulating mesenchymal stem cells (MSC) to increase osteogenesis, we evaluated the expression of bone-specific promoters during MSC differentiation. Multi-lineage potential of cultured MSC was confirmed by osteogenic, adipogenic and chondrogenic differentiation under controlled conditions. Recombinant adeno-associated virus (rAAV) encoding luciferase under the human cytomegalovirus (CMV), mouse alkaline phosphatase (ALP), Runx-2/cbfa1 (RUNX), osteopontin (OPN), collagen type 1a (COL), and osteocalcin (OCN) promoters was used to transduce mouse MSC. Replicate cultures were maintained undifferentiated or differentiated to osteoblast lineage. Luciferase expression was determined on days 1, 2, 3, 7, 14, or 21 as a measure of promoter activity. Expression of osteogenic markers and mineralization was determined as correlates of osteopoiesis. Results indicated expression from CMV promoter in undifferentiated and differentiated cultures at early stage. However, expression from COL and RUNX promoters was abundant only in differentiating cultures as early as 24 h but declined gradually. Expression from OPN and ALP promoters was evident 24 h following osteogenic differentiation and peaked gradually until 2 weeks before declining. Expression from OC promoter was evident only after 7 days of differentiation but remained until final analysis on day 21. That rAAV transduction of MSC does not induce differentiation was also confirmed by quantitative reverse-transcription polymerase chain reaction (QRT-PCR). The observed stage-specific expression of analyzed promoters was not significant when the MSC were differentiated to adipocytes. Thus, the use of RUNX2 or COL promoter to stably express osteoinductive factors in MSC may allow both self-renewal of modified MSC and enrichment of osteoblast commitment.