Macrophages encode several Toll-like receptors (TLRs) that recognize bacterial components, such as lipoproteins (TLR2) or lipopolysaccharides (TLR4), and activate multiple signaling pathways. Activation of transcription factor NF-κB by TLR2 or TLR4 signaling promotes proinflammatory and cell survival responses. Alternatively, TLR2 or TLR4 signaling can promote apoptosis if the activation of NF-κB is blocked. The gram-negative bacterial pathogen Yersinia pseudotuberculosis secretes into macrophages a protease (YopJ) that inhibits the activation of NF-κB and promotes apoptosis. We show that primary macrophages expressing constitutively active inhibitor κB kinase β (IKKβ) are completely resistant to YopJ-dependent apoptosis, indicating that YopJ inhibits signaling upstream of IKKβ. Apoptosis is reduced two- to threefold in TLR4−/− macrophages infected with Y. pseudotuberculosis, while the apoptotic response of TLR2−/− macrophages to Y. pseudotuberculosis infection is equivalent to that of wild-type macrophages. Therefore, TLR4 is the primary source of apoptotic signaling in Yersinia-infected macrophages. Our results also show that a small percentage of macrophages can die as a result of an apoptotic process that is YopJ dependent but does not require TLR2 or TLR4 signaling.