A diverse set of innate immune mechanisms protects cells from viral infections. The APOBEC3 family of DNA cytosine deaminases is an integral part of these defenses. For instance, APOBEC3D, APOBEC3F, APOBEC3G, and APOBEC3H would have the potential to destroy HIV-1 complementary DNA replication intermediates if not for neutralization by a proteasomal degradation mechanism directed by the viral protein Vif. At the core of this complex, Vif heterodimerizes with the transcription cofactor CBF-b, which results in fewer transcription complexes between CBF-b and its normal RUNX partners. Recent studies have shown that the Vif/CBF-b interaction is specific to the primate lentiviruses HIV-1 and SIV (simian immunodeficiency virus), although related nonprimate lentiviruses still require a Vif-dependent mechanism for protection from host species’ APOBEC3 enzymes. We provide a molecular explanation for this evolutionary conundrum by showing that CBF-b is required for expression of the aforementioned HIV-1–restrictive APOBEC3 gene repertoire. Knockdown and knockout studies demonstrate that CBF-b is required for APOBEC3 mRNA expression in the nonpermissive T cell line H9 and in primary CD4+ T lymphocytes. Complementation experiments using CBF-b separation-of-function alleles show that the interaction with RUNX transcription factors is required for APOBEC3 transcriptional regulation. Accordingly, the infectivity of Vif-deficient HIV-1 increases in cells lacking CBF-b, demonstrating the importance of CBF-b/RUNX–mediated transcription in establishing the APOBEC3 antiviral state. These findings demonstrate a major layer of APOBEC3 gene regulation in lymphocytes and suggest that primate lentiviruses evolved to hijack CBF-b in order to simultaneously suppress this potent antiviral defense system at both transcriptional and posttranslational levels.