Adipose-derived mesenchymal cells (AMCs) offer great promise for tissue engineering of bone. Previously, 1,25-dihydroxyvitamin D3, retinoic acid (RA), and dexamethasone had been shown to promote osteogenesis in bone marrow-derived mesenchymal cells (BMSCs). To study the osteogenic characteristics of mouse AMCs, we applied these 3 hormones alone and in combination to the AMCs and examined markers of osteogenic differentiation. Interestingly, vitamin D and RA demonstrated a consistent, dose-dependent enhancement of osteogenesis and upregulated osteoblast specific markers including osteopontin and osteocalcin. However, in AMCs, dexamethasone clearly inhibited osteogenic differentiation in a dose dependent fashion and greatly increased the adipogenic marker peroxisome proliferator activated receptor gamma (PPAgamma). In summary, we show in vitro that vitamin D and RA are potential candidates to serve as enhancers of osteogenesis of AMCs and may be incorporated into future cell-based strategies for bone tissue engineering.