SEK1 (MKK4/JNKK) is a mitogen-activated protein kinase activator that has been shown to participate in vitro in two stress-activated cascades terminating with the SAPK and p38 kinases. To define the role of SEK1 in vivo , we studied stress-induced signaling in SEK1 −/− embryonic stem and fibroblast cells and evaluated the phenotype of SEK1 −/− mouse embryos during development. Studies of SEK1 −/− embryonic stem cells demonstrated defects in stimulated SAPK phosphorylation but not in the phosphorylation of p38 kinase. In contrast, SEK1 −/− fibroblasts exhibited defects in both SAPK and p38 phosphorylation, demonstrating that crosstalk exists between the stress-activated cascades. Tumor necrosis factor α and interleukin 1 stimulation of both stress-activated cascades are severely affected in the SEK1 −/− fibroblast cells. SEK1 deficiency leads to embryonic lethality after embryonic day 12.5 and is associated with abnormal liver development. This phenotype is similar to c-jun null mouse embryos and suggests that SEK1 is required for phosphorylation and activation of c-jun during the organo-genesis of the liver.