Primate lentiviruses, including HIV-1, transduce terminally differentiated, nondividing myeloid cells; however, these cells are refractory to infection by gammaretroviruses such as murine leukemia virus (MLV). Here, we present evidence that a cellular restriction is the obstacle to transduction of macrophages by MLV. Neutralization of the restriction by Vpx, a primate lentiviral protein previously shown to protect primate lentiviruses from a macrophage restriction, rendered macrophages permissive to MLV infection. We further demonstrate that this restriction prevents transduction of quiescent monocytes by HIV-1. Monocyte-HeLa heterokaryons were resistant to HIV-1 infection, while heterokaryons formed between monocytes and HeLa cells expressing Vpx were permissive to HIV-1 infection. Encapsidation of Vpx within HIV-1 virions conferred the ability to infect quiescent monocytes. Collectively, our results indicate that the relative ability of lentiviruses and gammaretroviruses to transduce nondividing myeloid cells is dependent upon their ability to neutralize a cellular restriction.