Toll-like receptor 2 (TLR2) agonists induce a subset of TLR4-inducible proinflammatory genes, which suggests the use of differential signaling pathways. Murine macrophages stimulated with the TLR4 agonist Escherichia coli lipopolysaccharide (LPS), but not with TLR2 agonists, induced phosphorylation of signal transducer and activator of transcription I α (STATIα) and STATIβ, which was blocked by antibodies to interferon β (IFN-β) but not IFN-α. AII TLR2 agonists poorly induced IFN-β, which is encoded by an immediate early LPS-inducible gene. Thus, the failure of TLR2 agonists to induce STATI-dependent genes resulted, in part, from their inability to express IFN-β. TLR4-induced IFN-β mRNA was MyD88- and PKR (double-stranded RNA-dependent protein kinase)-independent, but TIRAP (Toll-interleukin I receptor domain-containing adapter protein)-dependent. Together, these findings provide the first mechanistic basis for differential patterns of gene expression activated by TLR4 and TLR2 agonists.