Achieving cell-specific expression of a therapeutic transgene by gene transfer vectors represents a major goal for gene therapy. To achieve specific expression of a transgene in CD4⁺ cells, we have generated lentiviral vectors expressing the enhanced green fluorescent protein (eGFP) reporter gene under the control of regulatory sequences derived from theCD4 gene—a minimal promoter and the proximal enhancer, with or without the silencer. Both lentiviral vectors could be produced at high titers (more than 10⁷ infectious particles per milliliter) and were used to transduce healthy murine hematopoietic stem cells (HSCs). On reconstitution of RAG-2–deficient mice with transduced HSCs, the specific vectors were efficiently expressed in T cells, minimally expressed in B cells, and not expressed in immature cells of the bone marrow. Addition of the CD4gene-silencing element in the vector regulatory sequences led to further restriction of eGFP expression into CD4⁺ T cells in reconstituted mice and in ex vivo–transduced human T cells. Non–T CD4⁺ dendritic and macrophage cells derived from human CD34⁺ cells in vitro expressed the transgene of the specific vectors, albeit at lower levels than CD4⁺ T cells. Altogether, we have generated lentiviral vectors that allow specific targeting of transgene expression to CD4⁺ cells after differentiation of transduced mice HSCs and human mature T cells. Ultimately, these vectors may prove useful for in situ injections for in vivo gene therapy of HIV infection or genetic immunodeficiencies.