We have developed a lentiviral vector system for human factor IX (hFIX) gene transfer in hematopoietic stem cells (HSCs) that provides erythroid cell-derived systemic protein delivery following nonmyeloablative conditioning and in vivo methylguanine methyltransferase (MGMT) drug selection. After bone marrow transplantation under moderate Busulfan conditioning, the initial hFIX expression in the chimeras was minimally detectable. However, the hFIX levels rose sharply following in vivo MGMT-drug selection and eventually reached a level that is considered curative in hemophilia B therapy (>500 ng/ml). The rise of hFIX levels was proportional to the increase in vector copy (VC) number in peripheral blood cells. High levels of hFIX expression were maintained in serially engrafted mice chimeras for 18 months. Importantly, high-level hFIX expression by erythroid cells did not result in anemia or adversely affect red blood cell counts. The prospect of combining reduced intensity conditioning, a presumably lowered risk of insertional mutagenesis due to low VC number requirement and erythroid-restricted transgene expression, as well as long-term protein expression at high level, strongly supports the potential applicability of adult stem cell-based gene therapy in nonlethal blood or metabolic disorders, as demonstrated here for hemophilia.