Abstract: Objective To investigate the effectiveness of in vitro expansion of human umbilical cord hematopoietic stem cells (HSCs) and to optimize the culture conditions. Methods HSCs were cocultured with osteoblasts in the three-direction (3D) culture system or condition medium (CM) to study the expansion effect on HSCs. Results In the conditions of cocultured with osteoblasts and added with 15%CM produced an expansion of the HSCs at a higher level , the HSCs were evaluated that expansion 6.46-fold after 7th days incubation while 14.82-fold after 14th days incubation. At the same conditions, the number of colony forming units of granulocyte and macrophage (CFU-GM) and erthroid burst-forming unit (CFU-E) were also significant higher than that of other groups. Conclusions The HSCs were cocultured with osteoblast in 15% CM medium not only to achieve admirable results for in vitro expansion HSCs but also sustain the self-renew capacity.