Engaging mammalian Toll-like receptors (TLRs) activate both the NF-κB and mitogen-activated protein kinase signaling pathways. Here we establish that mitogen-activated protein 3 kinase Tpl2, levels of which are markedly reduced innfkb1−/−cells, is required for extracellular signal-regulated kinase (ERK) activation in bone marrow-derived macrophages and B cells stimulated with diverse TLR ligands. Despite rescuing TLR-dependent ERK activation innfkb1−/−bone marrow-derived macrophages by using an estrogen receptor-regulated version of the mitogen-activated protein 3 kinase, c-Raf (Raf:ER), CpG or LPS induction of IL-10 was only partially restored innfkb1−/−cells expressing Raf:ER, a finding consistent with NF-κB1 regulating IL-10 by a combination of ERK-independent and -dependent mechanisms. Collectively, our findings indicate that the Tpl2/MEK/ERK signaling module is a master regulator of ERK-dependent gene expression downstream of TLRs in different hemopoietic cells.