The TLR5 agonist flagellin induces innate and adaptive immune responses in a MyD88-dependent manner and is under development as a vaccine adjuvant. In vitro studies indicate that, compared with other bacteria-derived adjuvants, flagellin is a very potent activator of proinflammatory gene expression and cytokine production from cells of nonhemopoietic origin. However, the role of nonhemopoietic cells in promoting flagellin-induced immune responses in vivo remains unclear. To investigate the relative contributions of the nonhemopoietic (radioresistant) and the hemopoietic (radiosensitive) compartments, we measured both innate and adaptive immune responses of flagellin-treated MyD88 radiation bone marrow chimeras. We observed that radiosensitive and radioresistant cells played distinct roles in the innate response to flagellin, with the radiosensitive cells producing the majority of the TNF-alpha, IL-12, and IL-6 cytokines and the radioresistant cells most of the KC, IP-10, and MCP-1 cytokines. Direct activation of either compartment alone by flagellin initiated dendritic cell costimulatory molecule up-regulation and induced a significant humoral immune response to the protein itself as well as to coinjected OVA. However, robust humoral responses were only observed when MyD88 was present in both cell compartments. Further studies revealed that hemopoietic and nonhemopoietic expression of the cytokines TNF-alpha and IL-6, but not IL-1, played an important role in promoting flagellin-induced Ab responses. Thus, in vivo both radioresistant and hemopoietic cells play key nonredundant roles in mediating innate and adaptive immune responses to flagellin.