Globin gene therapy requires abundant numbers of highly engraftable, autologous hematopoietic stem cells expressing curative levels of β-globin on differentiation. In this study, CD34⁺ cells from 31 thalassemic patients mobilized with hydroxyurea+granulocyte colony-stimulating factor (G-CSF), G-CSF, Plerixafor, or Plerixafor+G-CSF were transduced with the TNS9.3.55 β-globin lentivector and compared for transducibility and globin expression in vitro, as well as engraftment potential in a xenogeneic model after partial myeloablation. Transduction efficiency and vector copy number (VCN) averaged 48.4 ± 2.8% and 1.91 ± 0.04, respectively, whereas expression approximated the one-copy normal β-globin output. Plerixafor+G-CSF cells produced the highest β-globin expression/VCN. Long-term multilineage engraftment and persistent VCN and vector expression was encountered in all xenografted groups, with Plerixafor+G-CSF–mobilized cells achieving superior short-term engraftment rates, with similar numbers of CD34⁺ cells transplanted. Overall, Plerixafor+G-CSF not only allows high CD34⁺ cell yields but also provides increased β-globin expression/VCN and enhanced early human chimerism under nonmyeloablative conditions, thus representing an optimal graft for thalassemia gene therapy.