The atypical E3 ligase HOIL-1 forms ester bonds between ubiquitin and serine/threonine residues in proteins, but the physiological roles of this unusual modification are unknown. We now report that IL-18 signalling leading to the production of interferon γ (IFNγ) and granulocyte-macrophage colony-stimulating factor (GM-CSF) is enhanced in cytotoxic T cells from knock-in mice expressing the E3 ligase-inactive HOIL-1[C458S] mutant, demonstrating that the formation of HOIL-1-catalysed ester-linked ubiquitin bonds restricts the activation of this pathway. We show that the interaction of IRAK2 with TRAF6 is required for IL-18-stimulated IFN-γ and GM-CSF production, and that the increased production of these cytokines in cytotoxic T cells from HOIL-1[C458S] mice correlates with an increase in both the number and size of the Lys63/Met1-linked hybrid ubiquitin chains attached to IRAK2 in these cells. In contrast, the secretion of IL-12 and IL-6 and the formation of il-12 and il-6 mRNA induced in bone marrow-derived macrophages (BMDMs) by prolonged stimulation with TLR-activating ligands that signal via myddosomes, which also requires the interaction of IRAK2 with TRAF6, were not increased but modestly reduced in HOIL-1[C458S] BMDM. The decreased production of these cytokines correlated with reduced ubiquitylation of IRAK2. Our results establish that changes in HOIL-1-catalysed ester-linked ubiquitylation can promote or reduce cytokine production depending on the ligand, receptor and immune cell and may be explained by differences in the ubiquitylation of IRAK2.