The establishment of HIV proviral latency requires the creation of repressive chromatin structures that impair the initiation of transcription and restrict RNAP II elongation. We have found that C-promoter binding factor-1 (CBF-1), a CSL (CBF-1, Su(H) and Lag-1)-type transcription factor and key effector of the Notch signaling pathway, is a remarkably potent and specific inhibitor of the HIV-1 LTR promoter. Knockdown of endogenous CBF-1 using specific small hairpin RNAs expressed on lentiviral vectors results in the partial reactivation of latent HIV proviruses, recruitment of RNAP II, loss of histone deacetylases and the concomitant acetylation of histones. An important property of any repressor utilized to establish HIV latency is that it must become displaced or deactivated upon T-cell activation. Consistent with this hypothesis, CBF-1 mRNA and protein levels are highest in quiescent or unstimulated T cells but decline rapidly in response to proliferative stimulation such as activation of the T-cell receptor or treatment with TNF-alpha. We conclude that CBF-1 is a previously overlooked factor that induces transcriptional silencing during the establishment of HIV latency.