There is a need for a deeper understanding of the biochemical events affecting embryonic stem (ES) cell culture by analyzing the expansion of mouse ES cells in terms of both cell growth and metabolic kinetics. The influence of the initial cell density on cell expansion was assessed. Concomitantly, the biochemical profile of the culture was evaluated, which allowed measuring the consumption of important substrates, such as glucose and glutamine, and the production of metabolic byproducts, like lactate. The results suggest a more efficient cell metabolism in serum-free conditions and a preferential use of glutaminolysis as an energy source during cell expansion at low seeding densities. This work contributes to the development of fully-controlled bioprocesses to produce relevant numbers of ES cells for cell therapies and high-throughput drug screening.