Although hematopoietic cell gene therapy using retroviral vectors has recently achieved success in clinical trials, safety issues regarding vector insertional mutagenesis have emerged. Vector insertion, resulting in transcriptional activation of proto-oncogenes, played a role in the development of lymphoid leukemia in an X-linked severe combined immunodeficiency trial, and caused myeloid clonal dominance in a trial for chronic granulomatous disease. These events have raised the question of whether gene therapy for other disorders such as beta-thalassemia and sickle cell disease may hold a similar risk. In this study, we prospectively evaluated whether gamma-globin lentiviral vectors containing enhancer elements from the beta-globin locus control region could alter the expression of genes near the vector insertion. We studied this question in primary, clonal murine beta-thalassemic erythroid cells, where globin regulatory elements are highly active. We found an overall incidence of perturbed expression in 28% of the transduced clones, with 11% of all genes contained within a 600-kilobase region surrounding the vector-insertion site demonstrating altered expression. This rate was higher than that observed for a lentiviral vector containing a viral long-terminal repeat (LTR). This is the first direct evidence that lentiviral vectors can cause insertional dysregulation of cellular genes at a frequent rate.