HIV-1 entry into CD4+ T cells requires binding of the virus to CD4 followed by engagement of either the CCR5 or CXCR4 coreceptor. Pharmacologic blockade or genetic inactivation of either coreceptor protects cells from infection by viruses that exclusively use the targeted coreceptor. We have used zinc-finger nucleases to drive the simultaneous genetic modification of both ccr5 and cxcr4 in primary human CD4+ T cells. These gene-modified cells proliferated normally and were resistant to both CCR5- and CXCR4-using HIV-1 in vitro. When introduced into a humanized mouse model of HIV-1 infection, these coreceptor negative cells engraft and traffic normally, and are protected from infection with CCR5- and CXCR4-using HIV-1 strains. These data suggest that simultaneous disruption of the HIV coreceptors may provide a useful approach for the long-term, drug free treatment of established HIV-1 infections.