The extracellular signal-regulated kinase (ERK) group of MAPKs is essential for cell proliferation, including that stimulated by mitogens, oncogenic ras and raf. The Raf kinases (especially B-Raf) are ERK-specific, mitogen-activated MAP3Ks. Mixed lineage kinase-3 (MLK3) is a MAP3K previously thought to be a selective regulator of the JNK group of MAPKs. Surprisingly, we found that silencing of mlk3 by RNAi suppresses mitogen and cytokine activation not only of JNK but of ERK and p38 as well. Silencing mlk3 also blocks mitogen-stimulated phosphorylation of B-Raf at Thr598 and Ser601-a step required for B-Raf activation. Finally, silencing mlk3 prevents serum-stimulated cell proliferation and the proliferation of tumor cells bearing either oncogenic Ki-Ras or loss of function neurofibromatosis-1 (NF1) or NF2 mutations. The proliferation of tumor cells with activating mutations in B-raf or raf-1 are unaffected by silencing mlk3. These results define a new role for MLK3 in B-Raf activation, ERK signaling and cell proliferation. Accordingly, targeting MLK3 could be beneficial to the treatment of tumors with activated receptor tyrosine kinase or ras mutations, and to the treatment of NF1 or NF2 tumors.