Interaction of the chemokine receptor CXCR4 with its ligand SDF-1α (SDF) has been reported to play an important role in engraftment of hematopoietic stem cells (HSC) in the bone marrow (BM). However a critical requirement for CXCR4 in HSC engraftment is still controversial. It also remains unclear whether the effects that CXCR4 has on hematopoietic cell engraftment are related to enhanced homing of HSC to the bone marrow cavity, increased retention in marrow microenvironment or direct and indirect effects of CXCR4 stimulation on stem and progenitor cell proliferation, self-renewal and survival. To address these questions we have overexpressed CXCR4 in human cord blood CD34+ cells by transduction with an MSCV retroviral vector containing CXCR4 and eGFP (MIG-CXCR4). CXCR4 overexpressing cells were compared with control cells transduced with vectors expressing eGFP alone (MIG). CD34+eGFP+ cells were selected after transduction by flow cytometry sorting. We confirmed that CD34+ cells transduced with the MIG-CXCR4 vector demonstrated increased CXCR4 expression compared with MIG vector transduced controls (mean channel fluorescence for CXCR4 was 340±77.8 for MIG-CXCR4 transduced CD34+ cells compared with 142±37.1 for MIG transduced cells, n=8). MIG-CXCR4 transduced CD34+ cells demonstrated significantly enhanced chemotaxis to SDF in transwell migration assays (36±2% migration for MIG-CXCR4 vs. 20±4% migration for MIG transduced CD34+ cells to 100nM SDF-1, n=4, p=0.05). CD34+ cells transduced with MIG-CXCR4 demonstrated a 1.52±0.4 fold increase in expansion of total cell number compared with controls after 1 week of in vitro culture with growth factors (GF) [SCF (50ng/ml), TPO (100ng/ml), FL (100ng/ml), SDF (60ng/ml), n=3]. However, enhanced cellular expansion was not sustained on further GF culture. To evaluate the effect of CXCR4 overexpression on in vivo engraftment, CD34+ cells transduced with MIG-CXCR4 and MIG vectors were injected intravenously into sublethally irradiated NOD/SCID mice and human hematopoietic cell engraftment was evaluated after 6–8 weeks. MIG-CXCR4 transduced cells demonstrated significantly higher levels of engraftment with human CD45+ cells compared with MIG transduced cells (8±4.8% vs. 0.22±0.07% CD45+ cells in bone marrow, 1.3±0.9% vs. 0.2±0.09% CD45+ cells in spleen, and 1.8±1.0% vs. 0.3±0.25% CD45+ cells in peripheral blood for MIG-CXCR4 vs. MIG transduced cells, respectively, n=5). In addition, markedly higher levels of CD34+ cell engraftment was observed in the bone marrow of animals receiving MIG-CXCR4 vs. MIG transduced cells (1.7±1.0% vs. 0.06±0.03% CD34+ cells respectively, n=5). Consistent with this, the human CFC frequency in bone marrow of mice receiving MIG-CXCR4 transduced CD34+ cells was increased compared to mice receiving MIG transduced cells (31±0.5 CFC/100,000 cells vs. 5±3.2 CFC/100,000 cells, n=2,3, respectively). In conclusion, our results indicate that ectopic expression of CXCR4 in CD34+ cells results in enhanced engraftment of human hematopoietic cells and increased maintenance of hematopoietic stem and progenitor cells in the NOD/SCID mouse model. The effects of CXCR4 overexpression are considerably more prominent in vivo than in direct in vitro assays. It therefore appears that altered stem and progenitor cell homing and microenvironmental interaction, rather than direct signaling to HSC, may be responsible for enhanced CD34+ cell engraftment and maintenance following CXCR4 receptor overexpression.