Distal cis-regulatory elements play essential roles in the T lineage-specific expression of cytokine genes. We have mapped interactions of three trans-acting factors-NF-κB, STAT4, and T-bet-with cis elements in the Ifng locus. We find that RelA is critical for optimal Ifng expression and is differentially recruited to multiple elements contingent upon T cell receptor (TCR) or interleukin-12 (IL-12) plus IL-18 signaling. RelA recruitment to at least four elements is dependent on T-bet-dependent remodeling of the Ifng locus and corecruitment of STAT4. STAT4 and NF-κB therefore cooperate at multiple cis elements to enable NF-κB-dependent enhancement of Ifng expression. RelA recruitment to distal elements was similar in T helper 1 (Th1) and effector CD8+ T (Tc1) cells, although T-bet was dispensable in CD8 effectors. These results support a model of Ifng regulation in which distal cis-regulatory elements differentially recruit key transcription factors in a modular fashion to initiate gene transcription induced by distinct activation signals. © 2010 Elsevier Inc.