The multipotent differentiation and immunosuppression capability of mesenchymal stem cells (MSCs) make it attractive source for stem cell therapy to treat serious diseases, including neural system diseases and immune disorders. For large scale clinical applications, MSCs have to be expanded to produce sufficient quantity for multiple treatments. While conventional passaging is not appropriate for such a task, bioreactor can be used to expand MSCs more efficiently. Yet the efficacy and biosafety of expanded MSCs must be properly assessed before the expanded MSCs can be implanted. This review presented state-of-the-art in expanding MSCs focusing on the progress on the assessment of the efficacy and biosafety of in vitro expanded MSCs. Current obstacles were discussed and future research directions were outlined.