Emerging evidence suggests a role for eosinophils in immune regulation of T cells. Thus, we sought to determine whether human eosinophils may exert their effect via differential generation of Th1 and Th2 chemokines depending on cytokines in their microenvironment and, if so, to establish the conditions under which these chemokines are produced. Eosinophils cultured with TNF-α plus IL-4 had increased mRNA expression and protein secretion of the Th2-type chemokines, CCL17 (thymus and activation-regulated chemokine) and CCL22 (macrophage-derived chemokine). Conversely, the Th1-type chemokines, CXCL9 (monokine induced by IFN-γ) and CXCL10 (IFN-γ-inducible protein-10), were expressed after stimulation with TNF-α plus IFN-γ. Addition of TNF-α appeared to be essential for IFN-γ-induced release of Th1-type chemokines and significantly enhanced IL-4-induced Th2-type chemokines. Inhibition of NF-κB completely blocked the production of both Th1 and Th2 chemokines. Activation of NF-κB, STAT6, and STAT1 was induced in eosinophils by TNF-α, IL-4, and IFN-γ, respectively. However, there was no evidence for enhancement of these signaling events when eosinophils were stimulated with the combination of TNF-α plus IL-4 or TNF-α plus IFN-γ. Thus, independently activated signaling cascades appear to lead to activation of NF-κB, STAT1, and STAT6, which may then cooperate at the promoter level to increase gene transcription. Our data demonstrate that TNF-α is a vital component for eosinophil chemokine generation and that, depending on the cytokines present in their microenvironment, eosinophils can promote either a Th2 or a Th1 immune response, supporting an immunoregulatory role for eosinophils.