Alveolar macrophages (AMs) normally respond to lipopolysaccharide (LPS) by activating Toll-like receptor (TLR)-4 signaling, a mechanism critical to lung host defense against gram-negative bacteria such as Pseudomonas aeruginosa. Because granulocyte macrophage colony-stimulating factor (GM-CSF)–deficient (GM^−/−) mice are hyporesponsive to LPS, we evaluated the role of GM-CSF in TLR-4 signaling in AMs. Pulmonary TNF-α levels and neutrophil recruitment 4 h after intratracheal administration of Pseudomonas LPS were reduced in GM^−/− compared with wild-type (GM^+/+) mice. Secretion of TNF-α by AMs exposed to LPS ex vivo was also reduced in GM^−/− mice and restored in mice expressing GM-CSF specifically in the lungs (SPC-GM^+/+/GM^−/− mice). LPS-dependent NF-κB promoter activity, TNF-α secretion, and neutrophil chemokine release were reduced in AM cell lines derived from GM^−/− mice (mAM) compared with GM^+/+ (MH-S). Retroviral expression of PU.1 in mAM cells, which normally lack PU.1, rescued all of these AM defects. To determine whether GM-CSF, via PU.1, regulated expression of TLR-4 pathway components, mRNA and protein levels for key components were evaluated in MH-S cells (GM^+/+, PU.1^Positive), mAM cells (GM^−/−, PU.1^Negative), and mAMPU.1+ cells (GM^−/−, PU.1^Positive). Cluster of differentiation antigen-14, radioprotective 105, IL-1 receptor–associated kinase (IRAK)-M mRNA, and protein were dependent upon GM-CSF and restored by expression of PU.1. In contrast, expression of other TLR-4 pathway components (myeloid differentiation-2, TLR-4, IRAK-1, IRAK-2, Toll/IL-1 receptor domain containing adapter protein/MyD88 adaptor-like, myeloid differentiation primary-response protein 88, IRAK-4, TNF receptor–associated factor-6, NF-κB, inhibitor of NF-κB kinase) were not GM-CSF or PU.1-dependent. These results show that GM-CSF, via PU.1, enables AM responses to P. aeruginosa LPS by regulating expression of a specific subset of components of the TLR-4 signaling pathway.