Metabolic flux analysis (MFA) was pet formed on mouse embryonic stem cells cultured under three distinct differentiation conditions: classical embryoid body formation (EB), and on surfaces coated with either gelatin (GEL) or matrigel (MAT). MFA was based on 15 metabolic reactions and eight transport steps, and was carried out based on measurements of four substrates and/or metabolites: glucose. lactate, glutamine, and glutamate. Fluxes representing biomass production remained fairly constant for all three culture conditions with at most a 40% variation. In contrast, major temporal variations, up to 500%, were observed for all other major central metabolic fluxes across all culture conditions. Fluxes were compared to gene-expression patterns measured by microarray analysis. Particularly interesting is the correlation between the metabolic fluxes with expression patterns of the corresponding genes of the pyruvate to lactate reaction, whereby the genes for several isoforms of the lactate dehydrogenase enzyme were examined. The patterns of this flux were notably different in the EB cultures compared to the GEL and MAT cultures and reflected differences in oxygen and nutrient transport in EB vs. the GEL and MAT cultures. Another novel finding of this study is an event occurring between Days 4 and 5 of differentiation, which was identified by a notable change in both the metabolic fluxes and gene-expression patterns. This suggests that metabolic patterns can be used as effective beacons of changes in differentiating stem cells. Overall, and qualitatively, core metabolic fluxes, under the three culture conditions examined, correlated well with gene-expression patterns. (C) 2010 American Institute of Chemical Engineers Biotechnol. Prog., 26: 1222-1229, 2010