Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is an important therapy for both malignant and non-malignant diseases, however, recipients of allo-HSCT suffer from post-transplant infection and T-cell mediated graft-versus-host-disease (GVHD). Recent studies have demonstrated the role of toll-like receptors (TLRs) in recognizing pathogen associated molecular patterns on microbial surfaces during the innate immune response. We hypothesized that TLR expression in innate immune cells would play an important role in the development of the GVHD and that TLR-deficiency could result in less GVHD. We performed bone marrow transplants (BMTs) by lethally irradiating recipient mice and intravenously injecting T-cell depleted donor bone marrow with varying concentrations of donor T-cells to induce GVHD. To study TLR expression in transplant settings, cells were obtained at various time points from recipients of a syngeneic BMT or allogeneic BMT with or without GVHD. Cells were stained with TLR2 and TLR4 antibodies and analyzed using flow cytometry. TLR knockout studies were performed in an allogeneic setting using wild type donor T cells combined with donor bone marrow deficient in TLR1, TLR2, TLR4, and MyD88; mice were monitored daily for survival and weekly for the development of GVHD. By Day 29 post-transplant, all transplant settings showed decreased percentages of TLR2+ and TLR4+ myeloid and dendritic cells from the spleen, bone marrow, liver, and blood. At Day 14, percentages of TLR2+ dendritic cells spiked above wild-type values in all transplant settings, however percentages of TLR2+ myeloid cells, as well as of TLR4+ myeloid and dendritic cells, showed no such spike. In all cases, expression patterns did not differ significantly between allogeneic transplants with and without GVHD. Of the four TLR knockout transplants, only mice injected with MyD88 knockout bone marrow and wild type T-cells showed significant and early protection from GVHD morbidity and mortality. These results suggest variation in TLR expression post-transplant are not affected by GVHD, but are probably determined by parameters related to reconstitution and tissue damage from the BMT conditioning regimen. Results from the MyD88 knockout transplant demonstrate a role for the TLR pathway in the donor-derived innate immune system in inducing GVHD. The lack of GVHD protection in TLR1, TLR2 and TLR4 knockout transplants may be due to redundancy in the TLR pathway. MyD88, however, remains a key convergence point of the pathway and thus its deletion produces more profound GVHD protection.