Position effects limit the curative potential of gene transfer strategies for the hemoglobinopathies by inducing clonal variability of transgene expression. We evaluated the mitigating effects of the chicken hypersensitivity site 4 (HS4) insulator among lentiviral vector-transduced human hematopoietic cells. We constructed various lentiviral vectors using a green fluorescent protein (GFP) reporter under the control of a reverse-oriented murine stem cell virus (MSCV)-long-term repeat (LTR) promoter or a reverse-oriented β-globin expression cassette. A full-length HS4, a tandem HS4 core, and a single core insulator were inserted into the 3' LTR in both forward and reverse orientation. All but the reverse single core insulator significantly decreased titers. All reduced %GFP without increasing mean fluorescence intensity (MFI) among erythroid progeny of transduced human CD34(+) cells. A lower coefficient of variation (CV) was observed only among progeny of the full-length vector-transduced cells, yet a fivefold reduction in transduction efficiency was observed. In xenografted mice, the single core insulator decreased both the %GFP and the MFI at 4 and 8 weeks after transplantation with no difference in CVs. These data demonstrate that the inclusion of HS4 insulator elements lowers viral titers, reduces efficiency of transduction, and produces minimal effects on transgene expression among human hematopoietic cells in vitro and in vivo.