β-thalassemias are the most common single gene disorders and are potentially amenable to gene therapy. However, retroviral vectors carrying the human β-globin cassette have been notoriously unstable. Recently, considerable progress has been made using lentiviral vectors, which stably transmit the β-globin expression cassette. Thus far, mouse studies have shown correction of the β-thalassemia intermedia phenotype and a partial, variable correction of β-thalassemia major phenotype. We tested a lentiviral vector carrying the human β-globin expression cassette flanked by a chromatin insulator in transfusion-dependent human thalassemia major, where it would be ultimately relevant. We demonstrated that the vector expressed normal amounts of human β-globin in erythroid cells produced in in vitro cultures for unilineage erythroid differentiation. There was restoration of effective erythropoiesis and reversal of the abnormally elevated apoptosis that characterizes β-thalassemia. The gene-corrected human β-thalassemia progenitor cells were transplanted into immune-deficient mice, where they underwent normal erythroid differentiation, expressed normal levels of human β-globin, and displayed normal effective erythropoiesis 3 to 4 months after xenotransplantation. Variability of β-globin expression in erythroid colonies derived in vitro or from xenograft bone marrow was similar to that seen in normal controls. Our results show genetic modification of primitive progenitor cells with correction of the human thalassemia major phenotype.