Mesenchymal stem cells or marrow stromal cells (MSCs) represent a multipotent adult cellular population with immunomodulatory functions. In the adult human body, they are present in various niches, but their main source is bone marrow (BM). The regeneration capability of MSCs, their ease to undergo gene modification, as well as their immunosuppressive capacity render them as popular candidates for tissue engineering, gene therapy, and immunotherapy. They exhibit a unique in vitro expansion capacity, which however does not always compensate for the large number of cells required for cellular therapeutic applications. Unfortunately, to date, a uniform worldwide approach to MSC-culture is not available. Thus, in this chapter, we try to describe the optimal conditions for the successful isolation and ex vivo expansion of human MSCs from BM, to be used in all types of cellular therapeutic approaches. Moreover, we describe the methods for identification of their quality in terms of both multilineage potentiality and immunosuppressive ability. Detailed protocols for fetal calf serum selection and other culture parameters that affect the final outcome are described.