Lentiviral vectors derived from human immunodeficiency virus 1 (HIV-1) are increasingly used in the delivery of transgenes into mammalian cells for gene function studies and for potential treatment of human diseases. One of the major barriers of applying HIV vector in disease treatment is to provide sufficient amounts of infectious vectors at high concentration. HIV vectors were generated by transient transfection of human 293T cells with the packaging plasmids and the vector DNA. The exceptionally high transfection efficiency of this cell line leads to production of vector with titers ranging between 105 and 107 infectious particles/ml. We have shown that delivery of small hairpin RNA (shRNA) genes into human hematopoietic cells with HIV vectors led to efficient inhibition of HIV-1 replication. However, the presence of the anti-HIV shRNA gene resulted in at least a 10|[ndash]|100 fold reduction in vector titer. For efficient delivery of shRNA genes into human hematopoietic cells, methods to enhance vector production from 293T cells therefore need to be developed.