Abstract Recently, the search for stem cells has become focused on fetal appendages such as the amniotic membrane and umbilical cord. Previously, we have shown the existence of stem cells in the amniotic membrane that can differentiate into various cells. In this study, we attempt to characterize and maintain the stemness characteristics of mesenchymal stem cells (MSCs) for Wharton's jelly, an inherent tissue of the umbilical cord. Wharton's jelly cells (WJCs) were isolated, adhered to culture plates, and characterized for stem cell and surface markers expression. They expressed the embryonic stem cell markers Nanog, Oct ¾, and Sox2. On flow cytometric analysis, WJCs predominantly expressed the MSC markers CD73, CD90, and CD105 and did not express the hematopoietic lineage markers CD14, CD34, CD45, and HLA-DR. In floating culture, WJCs could maintain stemness, and they could differentiate to osteogenic, chondrogenic, and adipogenic lineages. In conclusion, WJCs satisfy the criteria of MSCs. Given that extraction of the umbilical cord is not invasive, and the umbilical cord can be obtained without ethical and technical issues, we suggest that WJCs, after maintaining stemness, have a potential contribution to medical treatment for patients, even newborns, with congenital skeletal and cartilage disorders.