Squamous cell carcinoma of the skin and head and neck region are a common and potentially fatal malignancy. Current chemotherapy treatments are generally not curative. Therefore, there is considerable need to develop new and targeted therapies to treat SCC. There is abundant data to show that E2Fs play an important role in many keratinocyte (KC) functions such as differentiation, growth and apoptosis. However, the identification of two new inhibitory forms of E2F, E2F7 and E2F8 has caused a rethink of our previous models. In this study, we examined the effects of E2F1, E2F7 and E2F8 on cytotoxic responses to chemotherapeutic agents. Our results showed that E2F8 deficient KCs displayed unaltered responses to 3 structurally different classes of chemotherapeutics represented by doxorubicin (anthracycline), cisplatin (platinum) or etoposide (topoisomerase II inhibitor). Similarly, E2F1 and E2F7-deficient KCs retained apoptotic responses to etoposide and cisplatin. In contrast, E2F7-deficient murine KCs were selectively sensitised to the cytotoxic actions of doxorubicin whilst E2F1-deficiency protected against doxorubicin. Moreover, sensitivity to doxorubicin was E2F7-dependent as reintroduction of E2F7 in E2F7-deficient KCs protected against doxorubicin-induced cell death. These results indicate that E2F7 regulate cytotoxic responses of SCC to doxorubicin, and doxorubicin toxicity is mediated, in part, by an E2F1-dependent mechanism. Since E2F7 modulates sensitivity to doxorubicin, we examined the cytotoxicity of doxorubicin in SCC cell lines. The results suggested that SCC cells vary in their sensitivity to doxorubicin and this correlates with the ratio of E2F7/E2F1 protein expression in SCC cells. These data showed that E2F7/E2F1 ratio is disrupted in SCC and this contributes to the insensitivity to doxorubicin. Combined, these data indicate the existence of a novel, non-redundant and isoform-specific activity of E2F7 in keratinocytes that has implications for our understanding of chemotherapeutic sensitivity in SCC.