We have recently shown that antigen presentation by recipient non-haematopoietic antigen presenting cells (APC) within MHC class II plays a major role in the initiation of GVHD of the GI tract. Here we focused on the mechanism of propagation and maintenance of GVHD within the context of indirect antigen presentation by donor APC. We used transgenic systems specific for recipient allogeneic peptides presented in MHC class I or class II in which donor antigen presentation could be spatially and temporally quantified in vivo. We found the mesenteric lymph node (mLN) to be the sentinel tissue for alloantigen presentation and T cell priming by donor APC during GVHD. The use of antibodies specific for alloantigen presentation within MHC and conditional deletion systems demonstrated that donor T cell priming was mediated by conventional donor CCR7+ CD11bnegCD103+ dendritic cells (DC) presenting alloantigen within the mLN. In the absence of CCR7, these DC remained in the colon but failed to migrate to the mLN and prime alloantigen specific T cells. In order to understand why this pathway was only operative in animals with GVHD and extensive gut injury, we analyzed the contribution of DAMP/PAMP pathways to this process. In the absence of MyD88/TRIF or RAGE, IL-12 secretion by mLN CD11bnegCD103+ DC was markedly attenuated and the absence of these molecules attenuated donor Th1 differentiation and expansion, an effect independent of alloantigen presentation. Interestingly, Th17 differentiation and expansion also occurred exclusively in the mLN in response to IL-6 from CD11b+CD103+ DC, again independent of concurrent antigen presentation. These data identify the mechanism by which DAMP/PAMP pathways act as the critical mediators of GVHD via IL-12 and IL-6. These cytokines represent important therapeutic targets since they drive T cell expansion and differentiation within the GI tract independently of alloantigen.