The stimulation of Toll-like receptors (TLRs) on macrophages triggers production of the cytokine tumor necrosis factor (TNF). TNF production occurs within 1 h of TLR stimulation and is sustained for 1 d. Here we document a function for the TNF family member 4-1BB ligand (4-1BBL) in sustaining TLR-induced TNF production. TLR signaling induced 4-1BBL, and 4-1BBL interacted with TLRs on the macrophage surface. The influence of 4-1BBL on TNF production was independent of its receptor (4-1BB) and did not require the adaptors MyD88 or TRIF. It did not influence TLR4-induced activation of transcription factor NF-kappaB (an early response) but was required for TLR4-induced activation of transcription factors CREB and C/EBP (a late event). Transient TLR4-MyD88 complexes appeared during the first hour after lipopolysaccharide stimulation, and TLR4-4-1BBL interactions were detected between 2 h and 8 h after lipopolysaccharide stimulation. Our results indicate that two different TLR4 complexes sequentially form and selectively control early and late TNF production.