In recent years, human mesenchymal stem cells (multipotential stromal cells) from bone marrow (hMSCs) have attracted enormous attention owing to their broad therapeutic potential. One of the problems in the overall therapeutic use of hMSCs has been the significant variability in the culture conditions used for their isolation and expansion. Since the seminal publications by Friedenstein and colleagues, the isolation and expansion of mesenchymal stromal cells (MSCs) from bone marrow have been of interest to several laboratories. As a result, numerous isolation protocols have been published. This chapter provides a simple protocol whereby a total of 80-100 million human MSCs, with an average viability greater than 90%, can be produced from a relatively small (1-3 mL) bone marrow aspirate in 14-20 days using double stacks culture chambers. MSCs were originally referred to as fibroblastoid colony forming cells because one of their characteristic features is adherence to tissue culture plastic and generation of colonies when plated at low densities. The efficiency with which they form colonies still remains an important assay for the quality of cell preparations. To assess the quality of cell preparations, two different colony forming unit (CFU) assays are also provided.