RUNX1 is frequently mutated in sporadic and inherited forms of hematologic malignancies, but the mechanism underlying it role in leukemogenesis remains poorly defined. In the first part of this work, we describe a novel role for RUNX1 in regulating TLR1/2 and TLR4 signaling and inflammatory cytokine production by neutrophils. Hematopoietic-specific RUNX1 loss increased the production of pro-inflammatory mediators, including tumor necrosis factor α (TNF-α), by bone marrow neutrophils in response to TLR1/2 and TLR4 agonists. Hematopoietic RUNX1 loss also resulted in profound damage to the lung following inhalation of the TLR4 ligand lipopolysaccharide. However, neutrophils with neutrophil-specific RUNX1 loss lacked the inflammatory phenotype caused by pan-hematopoietic RUNX1 loss, indicating that dysregulated TLR signaling is not due to loss of RUNX1 in neutrophils per se. Nevertheless, RUNX1-deficient neutrophils displayed broad transcriptional upregulation of many of the core components of TLR-mediated NF-κB signaling. Hence early, pan-hematopoietic RUNX1 loss de-represses an innate immune signaling transcriptional program that is maintained in terminally differentiated neutrophils, resulting in their hyper-inflammatory state. We hypothesize that inflammatory cytokine production by neutrophils may contribute to the disease progression in leukemia associated with RUNX1 mutations. In the second part of this work, we endeavor to understand the mechanism by which RUNX1 mutations cooperate with ASXL1 mutations in leukemia, given their significant co-occurrence in AML patients. We demonstrate that concurrent loss of RUNX1 and ASXL1 is not sufficient to induce hematologic malignancy but rather results in a lethal phenotype that is sensitive to the environment in which the mice are housed. For most of the phenotypes examined, including the inflammatory neutrophil phenotype, mice with loss of RUNX1 and ASXL1 are indistinguishable from mice that only have loss of RUNX1. Although we propose that the driver of the lethal phenotype is inflammatory due to its environmental sensitivity, further work will be required to pinpoint the exact cause of death beyond excluding hematologic malignancy. Together the data presented in this work spotlight inflammation as a significant consequence of RUNX1 loss and highlight a novel and targetable inflammatory mechanism through which RUNX1 mutations may impact normal and malignant hematopoiesis.