Recently, human mesenchymal stem cells (MSCs) are attracting attention as a useful source for regenerative therapy. Controlled production of cell therapy requires the establishment and management of an accurate isolation, characterization and monitoring for quality assurance of developing MSCs mediated. In this study, we were confirmed maintenance of potency of isolated and cultured human umbilical cord (hUC)-MSCs during ex vivo expansion or after cryopreservation. Expression of their cell specific marker was analyzed by flow cytometry and the differentiation potency was confirmed by guided differentiation of adipocyte, osteocyte, chondrocyte and hepatocyte after expanding over 15 doublings in vitro. Safe production of developing a cell therapy was proved by testing for microbial, mycoplasma, endotoxin, and adventitious agents. Also stability of cells in cultivation, preservation and/or differentiation was determined chromosomal assay. In developing using hUC-MSCs, cells showed an accurate isolation and stable expansion in ex vivo condition. The results of several management assay showed that the stem cell marker expression of CD31, CD34 and CD45 were under 10%, however CD90 was over 90% by FACS analysis. Any contamination and mutation in all tests weren't detected in specific points for safe or stable production of hMC-MSCs. Also the proliferation and differentiation potency maintains during in vitro culture and after cryopreservation of hUC-MSCs. These results could be used as standard methods of maintenance of hUC-MSCs for cell therapy products and clinical application.