Human mesenchymal stem cells (hMSCs) are adult stem cells with multipotent capacities. The ability of hMSCs to differentiate into many cell types, as well as their high ex vivo expansion potential, makes these cells an attractive therapeutic tool for cell transplantation and tissue engineering. One source of hMSCs in adult individuals is the bone marrow, where they are immersed in the stroma at a low frequency. Isolation of these cells requires in vitro experimentation and characterization based on immunophenotyping or functional traits. Human bone marrow of healthy donors was aspirated from the iliac crest. Mononuclear cells were layered over the Ficoll-Paque density-gradient and plated in tissue cultures dish. Following removal of non adherent cells 1–4 days after the establishment of the culture, cells are maintained with periodic passages until a relatively homogeneous population is established. The identification of adherent cells was performed by flow cytometry analysis. The cells were analyzed for the expression of CD34 (Hematopoietic marker), CD11b (Compliment receptor), CD31 (Platelet-Cell Adhesion Molecule), CD45 (Leukocyte Common antigen), CD105 (Endoglin), and CD73 (SH3/4). The in vitro differentiation of hMSCs into osteoblast and adipocytes was also achieved. Results: At the third passage, hMSCs were CD34, CD11b, CD31, and CD45 negative because antigen expression was less than 5%, while they showed a high expression of CD105 and CD73. The differentiation of osteoblasts is determined by deposition of a mineralized extracellular matrix in the culture plates that can be detected with Alizarin Red. Adipocytes are easily identified by their morphology and staining with Oil Red. Discussion: All these data suggest that MSCs can be isolated and expanded from most healthy donors, providing for autologous source of stem cell transplantation. In addition we have defined we have defined culture conditions under which hMSCs can be amplified about 108-fold in 6 weeks.