Mesenchymal stem cells (MSC) are a population of multipotent cells that can differentiate into various lineages such as fibroblast, osteoblast, chondrocyte and adipocyte and can be maintained over extended periods in vitro. They are also known to have a supporting role in hematopoiesis and in clinical bone marrow engraftment, MSC can alleviate graft-versus-host disease and strongly enhance bone healing, and are used in various other cell engineering applications. Fibroblasts constitute the connective tissue and support epithelial cells. Although they have already been extensively investigated, it is not clear whether stem cells exist in vivo which specifically regenerate fibroblasts. MSCs are a potential candidate, because of their fibroblastic shape in vitro and their differentiation towards the mesodermal lineage. One of the goals of this study is to characterize the in vivo differentiation potential of MSC. Although MSC can be extracted, cultured and expanded efficiently, current available methods rely on in vitro culture for the isolation and expansion of mesenchymal stem cells, which has been shown to induce alterations compared to primary MSC. Transplantation of such in vitro expanded MSCs has failed to allow long-term engraftment. We aim to improve this by preconditioning the recipient with exhaustion of endogenous MSCs and transplantation of freshly isolated MSCs. The characterization of surface markers of MSCs has been performed for in vitro expanded cells and some of these markers are actually used for MSCs' studies in vivo even though they have never been validated on freshly isolated MSCs. We have critically evaluated several of these previously established markers on freshly isolated, MSC containing cell populations using FACS sorting and in vitro differentiation methods to calculate MSC frequencies. We describe in this report an improved method for isolation of MSCs which combines a new extraction protocol that allows 10 fold enrichment, followed by depletion of CD45+ cells which achieves another 10 fold enrichment, and finally a FACS sorting for candidate markers. CD73 was confirmed as a positive marker that allows an enrichment of 1.5 fold. However, CD44 and CD105 were found to be not expressed despite being widely used as positive markers for in vitro expanded MSC in several studies. They allowed 2 and 1.5 fold enrichment, respectively, when used on freshly isolated MSCs. In conclusion, we were able to enrich mesenchymal stem cells from 1 per 27'000 in the initial preparation to 1 per 85 cells, which corresponds to a total enrichment above 500 fold