Correction of blood genetic disorders requires permanent gene transfer into self renewing, hematopoietic stem cells (HSC), and regulation of transgene expression in specific cell lineages. HIV-derived lentiviral vectors are very effective in transducing rare, non-dividing stem cell populations without altering their long-term repopulation and differentiation capacity. We developed a strategy for transcriptional targeting' of lentiviral vectors based on replacing the viral LTR control elements with cell lineage specific, genomic control elements. An upstream enhancer (HS2) of the erythroidspecific GATA-l gene was cloned in a second-generation lentiviral vector to replace most of the U3 region of the LTR, immediately upstream of the HIV-l promoter. The modified LTR was used to drive the expression of a reporter gene (GFP), while a second gene (~LNGFR) was placed under the control of an internal, constitutive promoter to monitor cell transduction, or immunoselect transduced cells, independently from the expression of the targeted promoter. The vector was used to transduce cell lines, human CD34⁺ hematopoietic stem/progenitor cells, and murine bone marrow HSCs. Gene expression was analyzed in the differentiated progeny of transduced stem cells in vitro and in vivo, after transplantation into lethally irradiated co-isogenic (for murine cells) or NOD/SCID (for human cells) mice. The transcriptionally targeted HIV LTR allowed very high level of transgene expression specifically in mature erythroblasts, in a <i>tat</i>-independent fashion and with no alteration in titer, infectivity, and genomic stability of the lentiviral vector. Expression from the targeted LTR was higher, better restricted, and showed significantly less position effect variegation than that obtained by the same combination of enhancer/promoter elements placed in the conventional, internal position. Cloning of the woodchuck hepatitis virus post transcriptional regulatory element (WPRE) at a defined position in the targeted vector, allowed selective accumulation of the genomic with respect to the internal RNA transcript, with no loss of cell-type restriction. A critical advantage of this targeting strategy is the use of the spliced, major viral transcript to express a therapeutic gene, and that of an internal, independently regulated promoter to express an additional gene for either cell marking or <i>in vivo</i> selection purposes.