Tumor necrosis factor (TNF) is an important modulator of the innate immune system, responsible for the activation of immune cells as well as the removal of damaged cells. Aberrant TNF signalling can cause a number of pathologic conditions. TNF binding to TNF receptor 1 triggers the activation of nuclear factor kappa B (NF-κB) leading to pro-inflammatory gene expression as well as the activation of the caspase cascade leading to death. In this study we observed that knockdown or knockout of the ubiquitin E3 ligase, Pellino3, sensitizes cells to TNF-induced apoptosis. Suppressed expression of Pellino3 leads to the activation of NF-κB, thereby inducing the expression of a series of antiapoptotic proteins, indicating that the increased apoptosis that is observed in Pellino3-knockdown or –deficient cells is not due to the regulation of the NF-κB pathway. We found that Pellino3 deficiency enhanced formation of the death-induced signalling complex in response to TNF. Pellino3 directly interacts with DISC components, RIP1 and caspase-8, but not FADD. Furthermore we show that Pellino3 regulates the formation of DISC and apoptosis in a manner that is dependent on the FHA domain but independent of the RING-like domain of Pellino3. The physiological importance of Pellino3 as a regulator of TNF signalling is confirmed by Pellino3-deficient mice showing increased sensitivity to TNF-induced apoptosis and greatly increased lethality in response to TNF administration. These findings define Pellino3 as a novel regulator of TNF signalling and a critical determining factor in dictating whether TNF induces cell survival or death.