Endotoxin tolerance is a complex phenomenon characterized primarily by decreased production of proinflammatory cytokines, chemokines, and other inflammatory mediators, whereas the expression of other genes are induced or unchanged. Endotoxin tolerance is induced by prior exposure of murine macrophages/human monocytes, experimental animals, or people to TLR ligands. Although recent studies reported a possible relationship between endotoxin tolerance and differentiation of alternatively activated macrophages (AA-MΦs or M2), we show in this study that LPS pretreatment of IL-4Rα(-/-) and STAT6(-/-) macrophages, which fail to develop into AA-MΦs, resulted in tolerance of proinflammatory cytokines, as well as molecules and chemokines previously associated with AA-MΦs (e.g., arginase-1, mannose receptor, CCL2, CCL17, and CCL22). In contrast to LPS, wild-type (WT) MΦs pretreated with IL-4, the prototype inducer of AA-MΦs, did not induce endotoxin tolerance with respect to proinflammatory cytokines, AA-MΦ-associated chemokines, negative regulators, NF-κB binding and subunit composition, and MAPKs; conversely, IL-13(-/-) macrophages were tolerized equivalently to WT MΦs by LPS pretreatment. Further, IL-4Rα deficiency did not affect the reversal of endotoxin tolerance exerted by the histone deacetylase inhibitor trichostatin A. Like WT mice, 100% of LPS-tolerized IL-4Rα-deficient mice survived LPS + d-galactosamine-induced lethal toxicity and exhibited decreased serum levels of proinflammatory cytokines and AA-MΦ-associated chemokines induced by LPS challenge compared with nontolerized mice. These data indicate that the signaling pathways leading to endotoxin tolerance and differentiation of AA-MΦs are dissociable.