Mesenchymal stem cells (MSCs) possess great potential for use in regenerative medicine. However, their clinical application may be limited by the ability to expand their cell numbers in vitro while maintaining their differential potentials and stem cell properties. Thus the aim of this study was to test the effect of a range of medium supplements on MSC self-renewal and differentiation potential. Cells were cultured until confluent and subcultured continuously until reaching senescence. Medium supplementation with fibroblast growth factor (FGF)-2, platelet-derived growth factor (PDGF)-BB, ascorbic acid (AA), and epidermal growth factor (EGF) both increased proliferation rate and markedly increased number of cell doublings before reaching senescence, with a greater than 1,000-fold increase in total cell numbers for AA, FGF-2, and PDGF-BB compared with control cultures. Long-term culture was associated with loss of osteogenic/adipocytic differentiation potential, particularly with FGF-2 supplementation but also with AA, EGF, and PDGF-BB. In addition FGF-2 resulted in reduction in expression of CD146 and alkaline phosphatase, but this was partially reversible on removal of the supplement. Cells expressed surface markers including CD146, CD105, CD44, CD90, and CD71 by flow cytometry throughout, and expression of these putative stem cell markers persisted even after loss of differentiation potentials. Overall, medium supplementation with FGF-2, AA, EGF, and PDGF-BB greatly enhanced the total in vitro expansion capacity of MSC cultures, although differentiation potentials were lost prior to reaching senescence. Loss of differentiation potential was not reflected by changes in stem cell surface marker expression.