Cellular metabolism is intricately regulated by substrate availability, enzymatic activity, and compartmentalization within subcellular localization. It is also highly flexible, with cells demonstrating the ability to shift between metabolic pathways to maintain their function. Aberrant metabolic processes are linked to numerous diseases including obesity, diabetes, and cancer and these diseases often shows shifts from traditional homeostatic metabolism to alternative metabolic processes. Understanding the regulation of and flexibility in critical metabolic pathways is required for efficacious targeting of such pathways in disease states. The metabolite acetyl-CoA is involved in multiple metabolic pathways that are altered in disease. Acetyl-CoA metabolism is regulated, in part, by the subcellular localization of the enzymes that perform is synthesis and utilization. Nuclear-cytosolic acetyl-CoA is used for lipid synthesis and acetylation, processes that are often dysregulated in cancer. For this reason, the enzymes that produce it are attractive therapeutic targets, but flexibility in acetyl-CoA metabolism poses a problem for inhibiting these enzymes. A complete understanding of nuclear-cytosolic acetyl-CoA metabolism in order to identify proper strategies for targeting this pathway. Therefore, we utilize an approach to genetically ablate the known nuclear-cytosolic acetyl-CoA producers, ATP-citrate lyase (ACLY) and acyl-CoA synthetase short chain family member 2 (ACSS2), to probe alternate compensatory pathways and asses combinatorial treatments in a hepatocellular carcinoma model. We identify the acetylcarnitine shuttle as a previously uncharacterized link between mitochondria and nuclear-cytosolic acetyl-CoA that can regulate de novo lipogenesis and gene expression through histone acetylation. Moreover, we identify two potential nutrient conditions that may synergize with targeting of nuclear-cytosolic acetyl-CoA metabolism using genetic loss of ACLY in vivo and in vitro. Together, these findings have expanded our understanding of cellular acetyl-CoA metabolism and shed light on the possibility of successfully targeting these pathways in HCC.