Monophosphoryl lipid A (MLA), a nontoxic derivative of the endotoxin lipopolysaccharide (LPS), has been approved in the United States for use as a vaccine adjuvant. LPS and MLA are ligands of Toll-like receptor 4 (TLR4), and it has been unclear why LPS triggers toxic inflammation, whereas MLA generates safe and effective immunostimulation. Signaling downstream of TLR4 is mediated by the adaptor proteins TRIF [Toll-interleukin-1 (IL-1) receptor (TIR) domain-containing adaptor-inducing interferon-β], which is required for adaptive immune outcomes, and MyD88 (myeloid differentiation marker 88), which is responsible for many proinflammatory effects. Two models have provided nonexclusive explanations for the differential effects of LPS and MLA. According to the first model, MLA fails to induce maturation of the proinflammatory cytokine IL-1β because it fails to activate caspase-1, which is required for the conversion of pro-IL-1β into its bioactive form. The second model suggests that MLA triggers unequal engagement of both of the signaling adaptor pathways of TLR4, such that signaling mediated by TRIF is largely intact, whereas signaling mediated by MyD88 is incomplete. We show that the TRIF-biased signaling that is characteristic of low-toxicity MLA explains its failure to activate caspase-1. Defective induction of NLRP3, which depends on MyD88, led to decreased assembly of components of the IL-1β-activating inflammasome required for the activation of preformed, inactive procaspase-1. In addition, we elucidated the contributions of MyD88 and TRIF to priming of the NLRP3 inflammasome and demonstrated that TRIF-biased TLR4 activation by MLA was responsible for the defective production of mature IL-1β.