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Efficient ex vivo/in vivo selection of genetically modified hematopoietic stem/progenitor cells (HPCs) and T lymphocytes could greatly improve several gene therapy strategies. We have previously reported that primary murine HPCs, transduced with a bicistronic retroviral vector, co-expressing the catalytic subunit of gamma-glutamylcysteine synthetase (gamma-GCSh) and eGFP, could be selected by l-buthionine-S,R-sulfoximine (BSO). Upon ex vivo transduction with a low, defined gene dosage and BSO selection, HPCs were able to repopulate the bone marrow of syngeneic myeloablated hosts, showing multi-lineage expression [Hum Gene Ther, 16 (2005), 711]. We now provide 'proof-of-principle' that the same strategy can be applied to the gene therapy of graft-vs.-host disease (GVHD) subsequent to allogeneic bone marrow transplantation (ABMT), and of chromosome X-associated chronic granulomatous disease (CGD). Transfer of the herpes simplex virus-thymidine kinase (HSV-Tk) 'suicide' gene into donor T lymphocytes is a potential method to control GVHD after ABMT. However, an efficient selection system is required to eliminate non-HSV-Tk-expressing T lymphocytes before administration to the patient. We now report that, upon transduction with a retroviral vector, co-expressing gamma-GCSh and eGFP, and subsequent selection by BSO, over 95% human T lymphocytes were found to express eGFP; moreover, upon transduction with a novel retroviral vector co-expressing gamma-GCSh and HSV-Tk, and subsequent BSO treatment, over 95% of T lymphocytes could be eliminated by ganciclovir. The efficacy of the gamma-GCSh-BSO selection strategy was then tested on an in vitro model of CGD. Upon transduction of gp91 (phox)-deficient PLBKO cells with a novel bicistronic retroviral vector co-expressing human gp91 (phox) and gamma-GCSh, exposure to BSO for 48 h eliminated most non-transduced cells, resulting in selection of gp91 (phox)-expressing cells, and reconstitution of NADPH oxidase activity.