The B-cell chronic lymphocytic leukemia (CLL)/lymphoma 11B (BCL11B) gene plays a critical role in T-cell differentiation and proliferation. However, little is understood about the role of BCL11B in human hematopoietic stem/progenitor cells. Small interfering RNA (siRNA)-mediated suppression of the BCL11B was shown to induce apoptosis in human T-cell acute lymphoblastic leukemia cells. To further characterize the role of BCL11B in hematopoietic stem/progenitor cells and assess the safety of siRNA-mediated targeted therapy, the in vitro differentiation and proliferation of CD34(+) cells after BCL11B-siRNA935 treatment were studied. CD34(+) cells were sorted from three cases of umbilical cord blood by the magnetic activated cell sorting technique, and the purity was identified by flow cytometry. BCL11B-siRNA935 was delivered into CD34(+) cells by nucleofection and the BCL11B expression level was analyzed by quantitative real-time polymerase chain reaction. Erythroid burst-forming units (BFU-E), granulocyte/macrophage colony-forming units (CFU-GM), and megakaryocyte colony-forming units (CFU-Meg) were assessed using BCL11B-siRNA935-treated CD34(+) cells by the methylcellulose semi-solid culture method. The BCL11B expression level in CD34(+) cells was significantly lower than that in Molt-4 cells and peripheral blood mononuclear cells from healthy individuals. An approximate one-fold reduction in the BCL11B mRNA level was observed 24 hours post-transfection with BCL11B-siRNA935. However, there was no significant difference on the colony formation ability of BFU-E, CFU-GM, and CFU-Meg for CD34(+) cells between the BCL11B-siRNA935-treated and mock-transfected groups (P > 0.05). BCL11B suppression by RNA interference had no significant influence on the differentiation and proliferation of CD34(+) cells. In conclusion, the BCL11B-siRNA935 used in this study may be safe, and BCL11B may be considered a new candidate for targeted gene therapy in T-cell malignancies.