Changes in environmental factors, diet, and genetics all influence metabolism, which is frequently dysregulated at the cellular and organismal levels in diseases such as metabolic syndrome, cancer, and inborn errors of metabolism. These maladies are often intertwined; for example, metabolic diseases such as obesity and inborn errors of metabolism such as fumarate hydratase deficiency can both increase the risk for developing certain cancers. One metabolic pathway frequently altered in disease is de novo lipogenesis (DNL). Aberrant DNL is believed to play a critical role in pathogenesis of cancer and non-alcoholic fatty liver disease (NAFLD), a manifestation of metabolic syndrome in the liver. DNL requires the metabolite, acetyl-CoA, which is predominantly synthesized in the cytosol and nucleus from the cleavage of citrate through the action of ATP-citrate lyase (ACLY). Consistent with its role in DNL, elevated levels or activity of ACLY is frequently observed in cancer and NAFLD. Therefore, I utilized a genetic loss-of-function approach coupled with metabolomic methods to investigate how abrogating ACLY impacts metabolism in proliferating cells and the liver. Unexpectedly, impairment of ACLY leads to metabolic compensation through ACSS2-dependent acetate usage at the cellular and tissue levels. Moreover, by depleting ACLY, we identify a link between dietary carbohydrate and microbiome-derived acetate in supporting hepatic DNL. These findings have revised our understanding of acetyl-CoA metabolism in cells, and how nutritional sources feed into this pathway in disease contexts.