In acute myeloid leukemia (AML), activating mutations in FLT3 are the most common genetic abnormality. Multiple FLT3 inhibitors have been developed, including the FDA-approved inhibitor gilteritinib. However, AML patients only respond to gilteritinib for approximately 6 months due to the emergence of drug resistance. While gilteritinib eliminates blasts in peripheral circulation, residual blasts in the bone marrow microenvironment are protected by cytokines and growth factors. Persistence of these residual cells represents early resistance to treatment. How these cells adapt to survive in the marrow microenvironment remains unclear, but over time resistant subclones resume growth and lead to relapsed disease. At relapse, many patients have intrinsic resistance mutations, what we term late resistance. In this study, we used a stepwise model that charts the temporal evolution of early to late gilteritinib resistance. To recapitulate early resistance, we cultured the FLT3-ITD+ AML cell lines, MOLM-14 and MV4;11, with exogenous microenvironmental ligands that allow cells to become resistant to gilteritinib in a ligand-dependent manner. After 7 weeks, all cultures with ligand resumed growth (early resistance), whereas cells without ligand never resumed growth. Following ligand withdrawal, the cells become transiently sensitive to gilteritinib but resumed growth after 2 months (late resistance). We comprehensively analyzed early and late resistance by integrating whole exome sequencing, CRISPR/Cas9 screening, proteomics, metabolomics, and small-molecule inhibitor screening. Early resistance is characterized by slowly dividing cells and metabolic reprogramming, particularly with respect to lipid metabolism. Early resistant cultures also became uniquely dependent on Aurora kinase B (AURKB) for survival. We then validated these findings in primary AML cells from patients (N=11) treated with gilteritinib and found that early resistant cells demonstrated reduced cell cycle and alterations in lipid metabolism. Gene expression analysis of sequential stromal cell samples from AML patients (N=13) pre- and post gilteritinib treatment showed an increase in lipid metabolism following gilteritinib treatment, indicating that the microenvironment is also dynamic and in crosstalk with neighboring AML cells. Primary early resistant AML cells also became dependent on AURKB signaling, and were exquisitely sensitive to the combination of AURKB inhibitors and gilteritinib. In contrast, late resistance is driven by an expansion of pre-existing NRAS mutant subclones, consistent with the resistance profile of AML patients on gilteritinib. Metabolic reprogramming continued to evolve in late resistance with further dependence upon lipid metabolism. Our study provides mechanistic understanding of how the marrow microenvironment contributes to extrinsic early resistance, which then leads to late intrinsic resistance. We also define a unique vulnerability to AURKB inhibitors in early resistance that may thwart the expansion of late resistant NRAS subclones. Citation Format: Sunil K. Joshi, Tamilla Nechiporuk, Daniel Bottomly, Paul Piehowski, Julie A. Reisz, Janét Pittsenbarger, Andy Kaempf, Sara J.C. Gosline, Yi-Ting Wang, Tao Liu, Cristina E. Tognon, Angelo D’Alessandro, Jeffrey W. Tyner, Shannon K. McWeeney, Karin D. Rodland, Brian J. Druker, Elie Traer. The AML microenvironment catalyzes a step-wise evolution to gilteritinib resistance [abstract]. In: Proceedings of the AACR Virtual Special Conference on the Evolving Tumor Microenvironment in Cancer Progression: Mechanisms and Emerging Therapeutic Opportunities; in association with the Tumor Microenvironment (TME) Working Group; 2021 Jan 11-12. Philadelphia (PA): AACR; Cancer Res 2021;81(5 Suppl):Abstract nr LT022.