Recent reports indicate that the activating transcription factor 5 (ATF5) is required for the survival of cancer cells but not for noncancer cells. However, the mechanisms by which ATF5 regulates genes and promotes cell survival are not clear. Using a cyclic amplification and selection of targets (CASTing) approach, we identified a novel ATF5 consensus DNA binding sequence. We show in C6 glioma and MCF-7 breast cancer cells that ATF5 occupies this sequence and that ATF5 activates reporter gene expression driven by this site. Conversely, reporter activity is diminished when ATF5 activity is blocked or when ATF5 expression is down-regulated by serum withdrawal. We further show that early growth response factor 1 (Egr-1), whose promoter contains two adjacent ATF5 consensus binding sites at a conserved promoter position in rat, mouse, and human, is targeted and regulated by ATF5 in C6 and MCF-7 cells. These data provide new insight on the mechanisms by which ATF5 promotes gene regulation and cancer-specific cell survival.