ZFHX1A is expressed in proliferating cells in the developing embryo, and in the present study we provide evidence that its expression is confined to proliferating cells through dependence on the Rb (retinoblastoma protein) family/E2F cell cycle pathway. Mutation of the Rb or E2F1 genes lead to induction of ZFHX1A mRNA, implying that the Rb-E2F1 repressor complex is important for repression of ZFHX1A. This repression is associated with recruitment of an E2F-Rb-histone deacetylase repressor complex to the promoter. A dominant-negative form of E2F1 inhibited ZFHX1A expression in p16INK4a- cells where Rb is constitutively hyperphosphorylated and inactive, suggesting that E2F can contribute to ZFHX1A transactivation in the absence of functional Rb. ZFHX1A is an E-box-binding transcription factor whose binding sites overlap with those bound by Snail1 and 2, and ZFHX1B/SIP1 (leading to at least partially overlapping function; for example, each of the proteins can repress E-cadherin expression). We found that expression of Snail1 and ZFHX1B/SIP1 is also regulated by E2Fs, but in contrast with ZFHX1A this regulation is Rb-family-independent. Snail2 expression was unaffected by either E2F or the Rb family. We propose that the differential effects of the Rb family/E2F pathway on expression of these E-box-binding proteins are important in maintaining their distinct patterns (and thus distinct functions) during embryogenesis.