Mixed-lineage protein kinase 3 (MLK3) is a member of the mitogen-activated protein (MAP) kinase kinase kinase group that has been implicated in multiple signaling cascades, including the NF-kappaB pathway and the extracellular signal-regulated kinase, c-Jun NH(2)-terminal kinase (JNK), and p38 MAP kinase pathways. Here, we examined the effect of targeted disruption of the murine Mlk3 gene. Mlk3(-/-) mice were found to be viable and healthy. Primary embryonic fibroblasts prepared from these mice exhibited no major signaling defects. However, we did find that MLK3 deficiency caused a selective reduction in tumor necrosis factor (TNF)-stimulated JNK activation. Together, these data demonstrate that MLK3 contributes to the TNF signaling pathway that activates JNK.