Umbilical cord blood (UCB) is well known to be a rich source of hematopoietic stem cells with practical and ethical advantages. Because mesenchymal stem cells (MSCs) from bone marrow have been regarded as good materials for cell/gene therapy as well as for tissue engineering because of their multidifferentiation potential, a number of trials have been undertaken to isolate MSCs from UCB. However, the results have been controversial, and little has become known about the effect of cryopreservation on the isolation of these stem cells. In this study, we examined the ability of cryopreserved UCB-derived cells to produce MSCs. Various culture conditions, including the seeding concentrations of cells and the media used, were investigated. We were able to obtain adherent cell populations after 3 to 5 weeks in our culture conditions from UCB-derived mononuclear cell fractions that had undergone cryopreservation for 0.1 to 5 years. These cells exhibited a fibroblast-like morphology and typical mesenchymal-like immunophenotypes. The results indicate that cryopreserved human UCB fractions can be used as an alternative source of MSCs for experimental and clinical applications as well as for tissue engineering.