C D34⁺ cells are nonpermissive to infection by HIV strains X4 and R5, despite the fact that many CD34⁺ cells express high levels of the viral receptor protein CD4 and the coreceptor CXCR4 on their surface. In these cells, the co-receptor CCR5 protein, which, like CXCR4, is a chemokine receptor, is detected mainly intracellularly. We hypothesized that CD34⁺ cells secrete CCR5-binding chemokines and that these factors interfere with HIV R5 interactions with these cells, possibly by binding CCR5 or by inducing its internalization. We found that human CD34⁺ cells and CD34⁺KIT⁺ cells, which are enriched in myeloid progenitor cells, expressed and secreted the CCR5 ligands RANTES, MIP-1α, and MIP-1β and that IFN-γ stimulated expression of these chemokines. In contrast, SDF-1, a CXCR4 ligand, was not detectable in the CD34⁺KIT⁺ cells, even by RT-PCR. Conditioned media from CD34⁺ cell culture significantly protected the T lymphocyte cell line PB-1 from infection by R5 but not X4 strains of HIV. Interestingly, the secretion of endogenous chemokines decreased with the maturation of CD34⁺ cells, although ex vivo, expanded megakaryoblasts still secreted a significant amount of RANTES. Synthesis of CCR5-binding chemokines by human CD34⁺ cells and megakaryoblasts therefore largely determines the susceptibility of these cells to infection by R5 HIV strains. We postulate that therapeutic agents that induce the endogenous synthesis of chemokines in human hematopoietic cells may protect these cells from HIV infection.J. Clin. Invest. 104:1739–1749 (1999).