In this study, we used a common procedure to assess the potential of mobilized peripheral blood (MPB) and umbilical cord blood (UCB) as sources of mesenchymal stem cells (MSCs) in comparison with bone marrow (BM). We tested three methods: plastic adhesion supplemented with 5% of BM-MSC conditioned medium, unsupplemented plastic adhesion, and selection of CD133-positive cells. MSCs derived from MPB or UCB are identified by their positive expression of mesenchymal (SH2, SH3) and negative expression of hematopoietic markers (CD14, CD34, CD45, HLA-DR). We observed that the CD133-positive cell fraction contains more MSCs with high proliferative potential. Placed in appropriate conditions, these cells proved their capacity to differentiate into adipocytes, osteocytes, chondrocytes, and neuronal/glial cells. MPB- and UCB-MSCs express Oct4, a transcriptional binding factor present in undifferentiated cells with high proliferative capacity. The selection of CD133-positive cells enabled us to obtain a homogeneous population of MSCs from UCB and MPB. These sources may have a major clinical importance thanks to their easy accessibility.