Thymic stromal lymphopoietin (TSLP) is a key cytokine that exacerbates allergic and fibrotic reactions. Several microbes and virus components have been shown to induce TSLP production, mainly in epithelial cells. TLR4 activators, such as lipopolysaccharide (LPS), induce TSLP production in vivo, although the underlying mechanisms remain unclear. In this study, we investigated the contribution of LPS-activated leukocytes to the production of TSLP in a mouse air-pouch-type inflammation model. LPS induced the production of TSLP in this model but not in the mouse keratinocyte cell line PAM212. Transfer of the infiltrated leukocytes collected from an LPS-injected air pouch to the air pouch of another mouse enhanced TSLP production. Further, the LPS-activated leukocytes produced tumor necrosis factor alpha (TNF-alpha) and interleukin-1 beta (IL-1 beta); a deficiency in these cytokines attenuated the LPS-induced production of TSLP. TSLP production was induced by TNF-alpha and enhanced by IL-1 beta and LPS in the PAM212 cells. These results demonstrated that TNF-alpha and IL-1 beta, which are partly produced by LPS-activated leukocytes, contribute to TSLP production via TLR4 activation in vivo.