The oncogenic retinoblastoma protein (Rb)/E2F pathway links cellular proliferation control to apoptosis as a fail-safe mechanism to protect aberrant oncogenic transformation. We have previously shown that histone deacetylase inhibitors (HDACIs) activate the E2F1-Bim apoptotic pathway, leading to efficient cell killing in cancer cells with deregulated E2F1 activity. To identify additional gene cassettes that might contribute HDACI-induced apoptosis upon E2F1 activation, we investigated the apoptotic transcriptional network affected by HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) in cancer cells with inducible E2F1. Data analysis focusing on 220 apoptosis-related genes identified apoptosis signal-regulating kinase 1 (ASK1) as one of a few genes in addition to Bim that are substantially up-regulated by SAHA upon E2F1 activation. We show that ASK1 is directly regulated by E2F1 and that prevention of ASK1 induction by RNA interference decreases SAHA-induced apoptosis. We further show that the role of ASK1 in the SAHA apoptotic response is not associated with its downstream effectors p38 or JNK. Instead, ASK1 knockdown results in reduced E2F1 transcriptional activity, leading to decreased Bim induction by SAHA. Moreover, ASK1 expression reverses the negative effect of Rb on E2F1 activity. These results indicate that ASK1 induction by E2F1 provides positive feedback regulation of E2F1 activity via Rb inhibition, which allows an efficient E2F1-Bim activation. Thus, the concomitant induction of E2F1 targets ASK1 and Bim by HDACIs warrants an effective activation of E2F1-dependent apoptosis in response to SAHA.