Conventionally, mesenchymal/stromal stem cells (MSC) are functionally isolated from primary tissue based on their capacity to adhere to the plastic surface. This isolation procedure is hampered by the unpredictable influence of secreted molecules or interactions with co-cultured hematopoietic and other unrelated cells as well as by the arbitrarily selected removal time of non-adherent cells prior to expansion of MSC. Early removal of non-adherent cells may result in the elimination of a late adhering MSC subsets and late removal increases the influence of undesired cells on the growth and differentiation of MSC. Finally, in conventional protocols MSC are co-expanded together with macrophages, endothelial cells and other adherent cells. To circumvent these limitations, several strategies have been developed to facilitate the prospective isolation of MSC based on the selective expression or absence of surface markers. Here we summarize the most frequently used markers and introduce new targets for antibody-based isolation procedures of primary bone marrow-derived MSC.