Homotrimeric TNF superfamily ligands signal by inducing trimers of their cognate receptors. As a biologically active heterotrimer, Lymphotoxin(LT)α₁β₂ is unique in the TNF superfamily. How the three unique potential receptor-binding interfaces in LTα₁β₂ trigger signaling via LTβ Receptor (LTβR) resulting in lymphoid organogenesis and propagation of inflammatory signals is poorly understood. Here we show that LTα₁β₂ possesses two binding sites for LTβR with distinct affinities and that dimerization of LTβR by LTα₁β₂ is necessary and sufficient for signal transduction. The crystal structure of a complex formed by LTα₁β₂, LTβR, and the fab fragment of an antibody that blocks LTβR activation reveals the lower affinity receptor-binding site. Mutations targeting each potential receptor-binding site in an engineered single-chain variant of LTα₁β₂ reveal the high-affinity site. NF-κB reporter assays further validate that disruption of receptor interactions at either site is sufficient to prevent signaling via LTβR.