CCR5 is the major HIV-1 co-receptor and its expression levels are a critical determinant of HIV-1 infection. However, the molecular mechanisms of CCR5 regulation in primary targets of HIV-1 remain unknown. Despite binding to conserved DNA-elements, we show that the transcription factors GATA-1 and GATA-3 differentially suppress the expression of CCR5 in stem cell-derived dendritic cells and primary human T cell subsets. In addition, GATA-1 expression was also more potent than GATA-3 in suppressing Th1 associated genes, IFNgamma and CXCR3. GATA-1, but not GATA-3, potently suppressed CCR5 transcription, thereby rendering human T cells resistant to CCR5-tropic HIV-1 infection. However, GATA-1 also substituted for GATA-3 in its canonical role of programming Th2 gene expression. These findings provide insight into GATA-3-mediated gene regulation during T cell differentiation. Importantly, decoding the mechanisms of GATA-1-mediated repression of CCR5 may offer an opportunity to develop novel approaches to inhibit CCR5 expression in T cells.