The CUL-4A ubiquitination machinery regulates the stability of HOXA9 by promoting its ubiquinitation and proteasome-dependent degradation. Our previous study showed the perturbation of CUL-4A biosysthesis in 32D cells by RNA-mediated interference increased an accumulation of HOXA9, and impaired G-CSF-induced terminal differentiation to granulocytes. HOXA9 is preferentially expressed in primitive hematopoietic cells and downregulates with differentiation. In this study, a lentiviral vector expressed both CUL-4A shRNA and GFP was constructed and was used to transduce cord blood (CB) CD34+ cells (GFP-shCUL-4A-CD34+ cells). The ability of GFP-shCUL-4A-CD34+ cells to form CFC colonies, to differentiate into myeloid, erythroid, B-lymphoid and T-lymphoid cells in vitro, and to engraft in irradiated NOD/SCID mice was evaluated. Our results showed that knockdown of CUL-4A m-RNA dramatically inhibited hematopoisis both in vivo and in vitro. In the CFC assay, FACS-sorted GFPbright-shCUL-4A-CD34+ cell formed fewer colonies (5%) that were smaller size (<500 cells/colonies) relative to progeny of GFPbright-CD34+ cells. In contrast, FACS-sorted GFPdim-shCUL-4A-CD34+ cells produced only 50% less colonies than the control GFPdim-CD34+ cells. However, GFPdim-shCUL-4A-CD34+ cells had 30% more colonies with <500 cells/colony but 82% less colonies with >500 cells/colony comparing to that of GFPdim-CD34+ cells. After transplanting the GFP-shCUL-4A-CD34+ cells into irradiated NOD/SCID mice for 6 weeks, GFP-shCUL-4A-CD45+ cells decreased more than 80% in the mouse bone marrow. These human GFP-shCUL-4A CD45 + cells were capable of forming GFP+-CFC but with >80% reduction in their potency. No reduction in % of GFP-CD45+ cells and GFP+-CFC was observed in the GFP-CD34+ cells group pre- and post-transplantation. The in vitro culture systems showed that knockdown of CUL-4A mRNA in CB CD34+ cells resulted in the developmental impairment in monocytic, megakaryocytic, erythroid, B lymphoid and T-lymphoid lineages. This impairment is a time- and shCUL-4A dose-dependent process. Further study using cell cycle analysis indicated that transduction of CUL-4A shRNA to HL-60 or CD34+ cells induced apoptosis. Our results, for the first time, demonstrate an essential role of the CUL-4A ubiquitin ligase in maintaining cell survival to ensure proper differentiation of normal human stem and progenitor cells into different hematopoietic lineages both in vivo and in vitro.