The aim of this study was to investigate the support effects of mesenchymal stem cells (MSCs) on umbilical cord blood (UCB) CD34+ cell (HSPC) expansion in vitro and its influence on cell characteristics including the surface marker of CD34+ cells, homing adhesion molecules and colony-forming ability. The mononucleated cells (MNCs) were isolated from UCB, then the CD34+ cells were isolated from freshly obtained MNCs by immunomagnetic beads, the MSC feeder cells exposed to gamma-ray of 137Cs were prepared by MSC feeder. The CD34+ cells were inoculated in different culture media. Experiment was divided into 3 groups: HSPC+CK group in which cytokines were added to medium (SCF, FL and TPO); HSPC+MSC group in which CD34+ cells were inoculated on MSC feeder; HSPC+MSC+CK group in which cytokines and MSC feeder cells were added to medium. After culture for 4, 7, 10, 14 days the MNC amount was counted and expansion ability of CD34+ cells was evaluated. The immunotypes of CD34+ cells and subsets, homing adhesion molecules and colony-forming ability in different groups detected by flow cytometry. The results showed that the amount of MNCs and CD34+ cells all obviously increased during culture for 14 days, the expansion levels of MNCs in 3 groups were HSPC+MSC+CK group>HSPC+CK group>HSPC+MSC group in proper order. Within 10 days of expansion in vitro amount of MNCs obtained significant expansion, meantime the expansion of CD34+ cells was higher also. The CD34+ count in 3 groups at day 4 of culture decreased significantly as compared with 0 day of culture (p<0.01). The CD34+ cells ratios in 3 groups after expansion were HSPC+MSC group>HSPC+MSC+CK group>HSPC+CK group in proper order (p<0.01), while CD34+ subset levels in 3 groups were different, the CD34+CD38- cells in HSPC+CK group at 4 days of culture increased transiently (62.71%), then quickly decreased, the CD34+CD38- cell ratio at day 7 was 0.05%, while the CD34+CD38- cell ratio in HSPC+MSC group at day 7 was 18.92%, difference was significant as compared with HSPC+CK group (p<0.05). The analysis of colony-forming units showed that the colony-forming ability at various time points after expansion all sustained in high level. It is concluded that in short-time (<7 day) culture of UCB CD34+ cells the combination of MSCs with cytokines can significantly expand the CD34+ cells and make the HSPCs to maintain original biologic characteristics.