The heat shock protein HSP27 protects cells against a wide variety of toxic treatments and blocks apoptosis induced by exposures to anticancer drugs and activation of the death receptor Fas. The molecular mechanisms of protection are unknown but appear to be regulated by phosphorylation of HSP27. Two apoptotic pathways can be activated downstream of Fas. The Fas-adaptor FADD mediates a caspase-dependent pathway. Fas also activates a caspase-independent pathway which correlates with Fas-induced translocation of Daxx from the nucleus to the cytoplasm and involves the interaction of Daxx with Fas and Ask1. We found that phosphorylated dimers of HSP27 interact with Daxx, preventing its interaction with Ask1 and Fas and blocking Daxx-mediated apoptosis. Expression of HSP27 also prevents the translocation of Daxx from the nucleus to the cytoplasm which is induced upon expression of Ask1 or stimulation of Fas. The observations reveal a new level of regulation of the Fas pathway. Whereas the FADD axis can be modulated by expression of FLIP, a natural inhibitor of FADD, our results show that HSP27 can accomplish a similar function for the Daxx axis.