There is an increasing interest in adult stem cells, especially mesenchymal stem/stromal cells (MSCs), in hematology and regenerative medicine because of the simplicity of isolation and ex vivo expansion of these cells. Conventionally, MSCs are functionally isolated from tissue based on their capacity to adhere to the surface of culture flasks. This isolation procedure is hampered by the unpredictable influence of secreted molecules and interactions with co-cultured hematopoietic and other unrelated cells, as well as by the arbitrarily selected removal time of non-adherent cells prior to the expansion of MSCs. Finally, functionally isolated cells do not provide biological information about the starting population. To circumvent these limitations, several strategies have been developed to facilitate the prospective isolation of MSCs based on the selective expression or absence of surface markers. The isolation and ex vivo expansion of these cells require an adequate quality control of the source and product. Here we summarize the most frequently used markers and introduce new targets for antibody-based isolation and characterization of bone marrow-derived MSCs.