The aim of this study was to evaluate whether human Wharton's jelly cells (WJCs) could be differentiated into nucleus pulposus (NP)-like cells by coculturing with NP cells (NPCs) in vitro. WJCs were isolated from the human umbilical cord, and NPCs were isolated from healthy human intervertebral disc. After coculturing WJCs with NPCs in a monolayer environment with or without cell-cell contact for 7 days, the real-time polymerase chain reaction showed the relative gene expressions of NP-marker genes (aggrecan, type II collagens, and SRY-type HMG box-9) were significantly increased (p<0.05) in all groups, and the increase in the group of 25:75/WJCs:NPCs was the largest (p<0.05). The increases of relative gene expression in WJCs cocultured with cell-cell contact were larger than those cocultured without contact in all ratios (p<0.05). WJCs were positive for telomerase expression. Flow cytometry analyses showed that WJCs expressed CD73, CD105, CD90, CD29, CD166, and human leukocyte antigen (HLA)-ABC while being negative for the expression of CD34, CD45, and HLA-DR. The results of this study indicated that the WJCs had the feature of the mesenchymal stem cell and might be induced to differentiate to NP-like cells by coculturing with NPCs.