IRAK4 is a central kinase in innate immunity but the role of its kinase activity is controversial. The mechanism of activation for IRAK4 is currently unknown; and little is known about the role of IRAK4 kinase in cytokine production, particularly in different human cell types. We show IRAK4 autophosphorylation occurs by an intermolecular reaction and that autophosphorylation is required for full catalytic activity of the kinase. Phosphorylation of any two of the residues T342, T345 and S346 are required for full activity and the death domain regulates the activation of IRAK4 . Using antibodies against activated IRAK4 we demonstrate that IRAK4 becomes phosphorylated in human cells following stimulation by IL-1R and TLR agonists, which can be blocked pharmacologically by a dual inhibitor of IRAK4 and IRAK1. Interestingly, in dermal fibroblasts, while complete inhibition of IRAK4 kinase activity does not inhibit IL-1-induced IL-6 production, NF-kB or MAP kinase activation, there is complete ablation of these processes in IRAK4 deficient cells. In contrast, the inhibition of IRAK kinase activity in primary human monocytes reduces R848 induced IL-6 production with minimal effect on NF-kB or MAP kinase activation. Taken together, these studies define the mechanism of IRAK4 activation; and highlight the differential role of IRAK4 kinase activity in different human cell- types as well as the distinct roles IRAK4 scaffolding and kinase functions play.