Macrophages and B-cells from Tpl2 knock-out mice exhibit a restricted defect in lipopolysaccharide and death receptor signaling that is limited to the activation of ERK. Here we show that Tpl2-/- MEFs exhibit defects in ERK, JNK, and NF-kappaB activation, or ERK activation only when stimulated with tumor necrosis factor-alpha (TNF-alpha) or interleukin-1beta, respectively. In addition, we show that the activation of Tpl2 by TNF-alpha depends on signals transduced by both TRAF2 and RIP1. Activated Tpl2 phosphorylates MKK4/SEK1 upstream of JNK and stimulates NF-kappaB DNA binding and transcriptional activity by mechanisms that are independent of the nuclear translocation of p50 and p65. Tpl2-transduced TNF-alpha signals instead promote the phosphorylation of p65 at Ser276 and modulate the spectrum of proteins associated with p65. Phosphorylation stimulates the transcriptional activity of NF-kappaB but does not affect its ability to bind DNA, which may be affected by the composition of the nuclear NF-kappaB complexes. These data confirm that defects caused by a single mutation may be cell-type and signal-specific and delineate the role of Tpl2 in the transduction of TNF-alpha signals that activate JNK and NF-kappaB in MEFs.