Abstract Fanconi anemia (FA) is the most common type of inherited bone marrow failure (BMF) syndromes and poses tremendous challenges in health care. The process of FA disease progression in the context of hematopoiesis is characterized by BMF, clonal proliferation of hematopoietic stem and progenitor cells (HSPCs), and progression to myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). While many studies have established a correlation between FA deficiency and defects in the HSC compartment, the mechanisms by which the FA proteins function in HSC maintenance remain largely unknown. During this past funding period, we have gathered evidence implicating pathogenic role of HSC-specific metabolic abnormality in FA. More recently, we identified the Fancd2-Atad3-Tufm complex using our newly developed Fancd23XFLAGHA knock- in model, and established a potential linkage of FA HSC failure to dysregulated mitochondrial translation and oxidative phosphorylation (OXPHOS). We hypothesize that the FANCD2/FA pathway restricts mitochondrial activity in HSC maintenance, and that loss of FA function leads to augmented mitochondrial translation and OXPHOS contributing to BMF and leukemic progression. The goals of the project are to investigate (1) the mechanism by which the FANCD2/FA pathway controls mitochondrial activity in HSC maintenance and (2) the link between augmented mitochondrial translation/OXPHOS and FA disease progression. To achieve these goals, we will first assess the functional consequence of the interaction between Fancd2 and the mitochondrial translational machinery by determining the structural elements of the biochemical interaction between Fancd2 and the Atad3-Tufm complex, the requirement for the Fancd2-Atad3-Tufm interaction in the control of mitochondrial translation and OXPHOS, and the functional link between the Fancd2-Atad3-Tufm interaction and HSC maintenance. We will then investigate whether there is a direct link between augmented mitochondrial translation/OXPHOS and FA disease progression in FA patients at three different stages (BMF, MDS, and AML), and the cellular mechanisms responsible for leukemic transformation in FA HSCs. Successful completion of the proposed study will not only improve mechanistic understanding of the interplay between the FA pathway and mitochondrial metabolism in the context of HSC maintenance, but also lead to a new avenue of research designed to target specific dysregulated metabolic checkpoints for developing innovative therapeutic strategies in FA and other BM failure and leukemia diseases.