Cord blood represents a large source of hematopoietic stem/progenitor cells. It can be induced to proliferate directly into erythroid progenitors in a appropriate ex vivo culture condition, then to generate mature red blood cells after injection into the body. The combination of Flt3 ligand, TPO, SCF and EPO is ideal for cord blood MNC to proliferate into erythroid progenitors. This study was aimed to evaluate the effect of FL, SCF and TPO on CD34(+) expansion. and to investigate influence of the cytokine combination on the proliferation of the CD34(+) cells. Mononuclear cells (MNC) were cultured in serum-free liquid culture system. Experiments were divided into 3 groups. In the group A as control no cytokines were added in the culture system; in the group B the cells cultured with SCF + FL + TPO + EPO + IGF-1; in the group C the cells were cultured with SCF + FL + TPO. EPO and IGF-1 were added at day 6. Part of the renewed MNCs and colony-forming units were counted, the proportion of CD34(+), CD34(+) CD71(+), CD71(+) GPA(+) cells was detected by FACS. The result showed that after 10 days, the the total cord blood cells increased 6.89-folds in group B and 3.06-folds in group C (P < 0.05). The CD34(+) cells increased 4.83-folds in group B and 2.47-folds in group C (P < 0.05). The colony-forming cells (CFCs) increased 4.3-folds in group B and 2.5-folds in group C (P < 0.05). Erythroid progenitors (BFU-E) and CFU-E increased 5.4-folds in group B and 3.1-folds in group C (P < 0.05). The CD34(+)CD71(+) cells increased 8.72-folds in group B and 3.37-folds in group C (P < 0.05). The CD71(+) GPA(+) cells increased 53.4-folds in group B and 30.29-folds in group C. They were different at any time point (P < 0.05). It is concluded that in the group with FL + SCF + TPO, CD34(+) cells and CFC can greatly be expanded from cord blood MNC in the serum-free culture system. In the group with FL + SCF + TPO + EPO + IGF-1, erythroid progenitors can greatly be expanded in the serum-free culture system, supplying EPO at day 0 was better than supplying at day 6. Since the largest number of colony-forming cells such as BFU-E and CFU-E were gained in the TPO + SCF + FL + EPO + IGF-1 group at day 10, the harvest time after cultivation in vitro should be selected at day 10.