Mesenchymal stem cells (MSCs), sometimes referred to as marrow stromal cells or multipotential stromal cells, represent a class of adult progenitor cells capable of differentiation to several mesenchymal lineages. They can be isolated from many tissues although bone marrow has been used most often. The MSCs may prove useful for repair and regeneration of a variety of mesenchymal tissues such as bone, cartilage, muscle, marrow stroma, and the cells produce useful growth factors and cytokines that may help repair additional tissues. There is also evidence for their differentiation to nonmesenchymal lineages, but that work will not be considered here. This chapter will provide the researcher with some background, and then provide details on MSC isolation, expansion and multilineage differentiation. These are the beginning steps toward formulating tissue repair strategies. The methods provided here have been used in many laboratories around the world and the reader can begin by following the methods presented here, and then test other methods if these prove unsatisfactory for your intended purpose.