Casein kinase 1 delta and epsilon (CK1δ/ɛ) are key regulators of diverse cellular growth and survival processes including Wnt signaling, DNA repair and circadian rhythms. Recent studies suggest that they have an important role in oncogenesis. RNA interference screens identified CK1ɛ as a pro-survival factor in cancer cells in vitro and the CK1δ/ɛ-specific inhibitor IC261 is remarkably effective at selective, synthetic lethal killing of cancer cells. The recent development of the nanomolar CK1δ/ɛ-selective inhibitor, PF670462 (PF670) and the CK1ɛ-selective inhibitor PF4800567 (PF480) offers an opportunity to further test the role of CK1δ/ɛ in cancer. Unexpectedly, and unlike IC261, PF670 and PF480 were unable to induce cancer cell death. PF670 is a potent inhibitor of CK1δ/ɛ in cells; nanomolar concentrations are sufficient to inhibit CK1δ/ɛ activity as measured by repression of intramolecular autophosphorylation, phosphorylation of disheveled2 proteins and Wnt/β-catenin signaling. Likewise, small interfering RNA knockdown of CK1δ and CK1ɛ reduced Wnt/β-catenin signaling without affecting cell viability, further suggesting that CK1δ/ɛ inhibition may not be relevant to the IC261-induced cell death. Thus, while PF670 is a potent inhibitor of Wnt signaling, it only modestly inhibits cell proliferation. In contrast, while sub-micromolar concentrations of IC261 neither inhibited CK1δ/ɛ kinase activity nor blocked Wnt/β-catenin signaling in cancer cells, it caused a rapid induction of prometaphase arrest and subsequent apoptosis in multiple cancer cell lines. In a stepwise transformation model, IC261-induced killing required both overactive Ras and inactive p53. IC261 binds to tubulin with an affinity similar to colchicine and is a potent inhibitor of microtubule polymerization. This activity accounts for many of the diverse biological effects of IC261 and, most importantly, for its selective cancer cell killing.