Cancer cells undergo intricate metabolic adaptations to thrive and survive amidst challenging conditions, particularly evident in solid tumors, where cells in the tumor core contend with severe hypoxia and nu-trient scarcity. However, the conventional 2D in vitro cancer models often fail to replicate these demand-ing conditions, offering a relatively uniform environment in terms of oxygen and nutrient exposure. This study aimed to delve into the significance of adipose triglyceride lipase (ATGL), a critical neutral lipid hydrolase, in cancer cells subjected to more tumor-like conditions. To achieve this objective, we cultivated 3D multicellular spheroids of ATGL-deficient lung cancer cells and subjected them to extensive proteomics analysis and metabolic phenotyping. Our findings reveal that the absence of ATGL boosted spheroid growth and facilitated their adaptation to hypoxia. This was accomplished by increasing glucose uptake, promoting a pro-Warburg effect, subsequently leading to alterations in lipid metabolism and heightened protein production. Intriguingly, this observed phenotype was further validated in a more "in vivo-like" setting when cancer spheroids were grown on the chick chorioallantoic membrane, but not when cells were cultured as a 2D monolayer. Furthermore, our analysis of publicly available cancer databases unveiled an inverse relationship be-tween ATGL expression and the increased reliance on glucose by cancer cells. In summary, our study presents compelling evidence that ATGL plays a pivotal role in influencing glucose metabolism in cancer cells when cultivated in 3D environments, mimicking the conditions of solid tumors. Lastly, our research underscores the necessity for alternative cell culture models, as many of the observed phenotypes in 3D and spheroids grown on the chick chorioallantoic membrane were absent in traditional 2D cell culture setups.