Hematopoietic stem cells are resistant to HIV-1 infection. Here, we report a novel mechanism by which the cyclin-dependent kinase inhibitor (CKI) p21^{Waf1/Cip1/Sdi1} (p21), a known regulator of stem cell pool size, restricts HIV-1 infection of primitive hematopoietic cells. Modifying p21 expression altered HIV-1 infection prior to changes in cell cycling and was selective for p21 since silencing the related CKIs, p27^Kip1 and p18^INK4C, had no effect on HIV-1. We show that p21 blocked viral infection by complexing with HIV-1 integrase and aborting chromosomal integration. A closely related lentivirus with a distinct integrase, SIVmac-251, and the other cell-intrinsic inhibitors of HIV-1, Trim5α, PML, Murr1, and IFN-α, were unaffected by p21. Therefore, p21 is an endogenous cellular component in stem cells that provides a unique molecular barrier to HIV-1 infection and may explain how these cells remain an uninfected “sanctuary” in HIV disease.