Components of bacteria and viruses activate Toll-Like Receptors in host cells, triggering the formation of the Myddosome and a signalling network that culminates in the production and release of the inflammatory mediators required to combat pathogenic infection. The Myddosome initiates signalling by recruiting and activating five E3 ligases that generate hybrid ubiquitin chains and attach them to components of the Myddosome. These ubiquitin chains act as a scaffold for the recruitment and activation of ubiquitin-binding proteins, which include the "master" protein kinases TAK1 and IKKβ that drive inflammatory mediator production, as well as other proteins like ABIN1 and A20 that restrict activation of the network to prevent the overproduction of these substances that can lead to autoimmunity and organ damage. Here we review recent developments in our understanding of this network, focusing on the unexpected discovery that the E3 ligase HOIL-1 initiates the formation of hybrid ubiquitin chains by forming an ester bond between the first ubiquitin and the protein components of the Myddosome.