Abstract Eukaryotic cells have established a robust system for regulating intracellular iron homeostasis based on the E3 ubiquitin ligase FBXL5 and its degradation of Iron Regulatory Proteins 1 and 2 (IRPs). In earlier funding periods, we established a paradigm in which FBXL5 acts as a signaling hub that integrates different physiological signals to coordinate the downstream IRP-mediated gene expression program. This application builds on that foundation to examine how signaling through the Fe-S cluster assembly, oxygen metabolism, and cell cycle pathways regulates the FBXL5-IRP axis. Specific aim 1 focuses on defining the mechanism by which the O2-dependent interaction of FBXL5 with the CIA targeting complex regulates IRP degradation. In specific aim 2, we test the hypothesis that degradation of the E3 ubiquitin ligase pVHL by FBXL5 regulates the response of cells to hypoxia. The experiments in specific aim 3 will examine the interplay between FBXL5 and cell cycle progression pathways to coordinate cell proliferation with metabolism. Together, these three aims will uncover the molecular mechanisms that govern how FBXL5 integrates and interprets signals transduced through multiple signaling pathways in order to dictate the multi-faceted cellular response to iron availability.