Lipids play important roles in biology that include structural compartmentation as membranes, energy storage, and regulatory functions as signaling molecules. These molecules can be obtained via the surrounding environment (e.g. diet) or synthesized de novo. Fatty acid synthesis is an energetically demanding process and must therefore be tightly regulated to balance fatty acid availability with the functional and energetic needs of cells and tissues. Here we review key aspects of de novo lipogenesis (DNL) in mammalian systems. We highlight key nodes in the pathway that are used for quantitation of lipogenic fluxes and regulation of fatty acid diversity across tissues. Next, we discuss key aspects of DNL function in the major lipogenic tissues of mammals: liver, white adipose tissue (WAT), and brown adipose tissue (BAT), highlighting recent molecular discoveries that suggest potential roles for tissue specific DNL. Finally, we propose critical questions that will be important to address using the advanced approaches for DNL quantitation described herein.