Faithful and efficient transmission of biological signals through mitogen-activated protein kinase (MAPK) pathways requires engagement of highly regulated cellular machinery in response to diverse environmental cues. Here, we report a novel mechanism controlling signal relay between two MAP3Ks, apoptosis signal-regulating kinase (ASK) 1 and ASK2. We show that ASK2 specifically interacts with 14-3-3 proteins through phosphorylated S964. Although a 14-3-3-binding defective mutant of ASK1 (S967A) has no effect on the ASK2/14-3-3 interaction, both overexpression of the analogous ASK2 (S964A) mutant and knockdown of ASK2 dramatically reduced the amount of ASK1 complexed with 14-3-3. These data suggest a dominant role of ASK2 in 14-3-3 control of ASK1 function. Indeed, ASK2 S964A-induced dissociation of 14-3-3 from ASK1 correlated with enhanced phosphorylation of ASK1 at T838 and increased c-Jun N-terminal kinase phosphorylation, the two biological readouts of ASK1 activation. Our results suggest a model in which upstream signals couple ASK2 S964 phosphorylation to the ASK1 signalosome through dual engagement of 14-3-3.