Tpl2/Cot is a serine/threonine kinase which plays a key physiological role in the regulation of immune responses to pro-inflammatory stimuli, including TNF-. TNF- stimulates the JNK, ERK and p38 mitogen-activated protein (MAP) kinases and the NF-B pathway by recruiting RIP1 and TRAF2 to the TNF receptor 1 (TNFR1). In the present study, we show that Tpl2 activation by TNF- signals depends on the integrity of the Tpl2-interacting proteins RIP1 and TRAF2 which are required for the engagement of the ERK MAP kinase pathway. However, neither RIP1 nor TRAF2 over-expression is sufficient to activate Tpl2 and ERK. We also show that Tpl2 activation by TNF- depends on a tyrosine kinase activity that is detected in TNF--stimulated cells. Based on both genetic and biochemical evidence, we conclude that in a variety of cell types, Syk is the tyrosine kinase which plays an important role in the activation of Tpl2 upstream of ERK. The presented data dissect the TNFR1 proximal events that regulate Tpl2 and ERK and highlight a role for RIP1, TRAF2 and Syk in this pathway.