We have previously described the development of oncoretrovirus vectors for human γ-globin using a truncated β-globin promoter, modified γ-globin cassette, and α-globin enhancer. However, one of these vectors is genetically unstable, and both vectors exhibit variable expression patterns in cultured cells, common characteristics of oncoretrovirus vectors for globin genes. To address these problems, we identified and removed the vector sequences responsible for genetic instability and flanked the resultant vector with the chicken β-globin HS4 chromatin insulator to protect expression from chromosomal position effects. After determining that flanking with the cHS4 element allowed higher, more uniform levels of γ-globin expression in MEL cell lines, we tested these vectors using a mouse bone marrow transduction and transplantation model. When present, the γ-globin cassettes from the uninsulated vectors were expressed in only 2% to 5% of red blood cells (RBCs) long term, indicating they are highly sensitive to epigenetic silencing. In contrast, when present the γ-globin cassette from the insulated vector was expressed in 49% ± 20% of RBCs long term. RNase protection analysis indicated that the insulated γ-globin cassette was expressed at 23% ± 16% per copy of mouse α-globin in transduced RBCs. These results demonstrate that flanking a globin vector with the cHS4 insulator increases the likelihood of expression nearly 10-fold, which in turn allows for γ-globin expression approaching the therapeutic range for sickle cell anemia and β thalassemia.