Mesenchymal stem cells MSCs were considered one of the most cells that contributed to research related to stem cells applications. And one of the most applied cells in cell therapy and clinical trials. Many sources were used to extract MSCs such as bone marrow, adipose tissue, pulp tissue, umbilical cord and cord blood. In this work cord blood was the source of cells due to its easy way to obtain and the easy way to process in the cell culture laboratory. The cord blood samples were processed inside safety cabinet class II to separate mononuclear cells first then the steps of isolation of MSCs comes later with culturing inappropriate media (DMEM, Fetal Bovine Serum FBS, L-Glutamine LG and penicillin-streptomycin) and conditions (temperature and CO2) to obtain pure mesenchymal stem cells after primary culture, subculture one and subculture two. The cells were characterized later with applied osteogenic differentiation to test MSCs capability to produce osteoblast. Cells show positive toward stains of calcium deposits of osteoblast (Von Kossa and Alizarine red S). The characterization also was achieved by using a flow cytometer to measure the reaction of MSCs toward the cell surface markers like CD 34, 45, 105 and 90. Cells show positive towards CD90 and 105 and negative toward CD 34 and 45. The methods applied for culturing and expansion mesenchymal stem cells in this work were suitable for yielding the number of the pure cell for further work. Also, the characterization methods (osteogenic differentiation, cell surface marker and morphology) were enough to identify mesenchymal stem cells.