Pennycress (Thlaspi arvense L.), a plant naturalized to North America, accumulates oil up to 30% of its seed dry weight. The fatty acid (FA) composition of pennycress oil was shown to be suitable for biodiesel and other industrial applications. However, for this plant to become an economically viable industrial crop, its oil production needs to be increased by metabolic engineering and/or breeding. In embryos, fatty acid synthesis (FAS) requires ATP, reductant, and carbon skeletons, which are provided by central metabolism. Thus, understanding relative contributions of these pathways can reveal potential targets for improving FAS in pennycress embryos. Towards this goal, we first combined untargeted and targeted metabolomics to identify the active pathways involved in FAS by providing ATP, reductant, and carbon skeletons. For this study, intracellular metabolites from embryos at 11, 13, 15, 17, 19 and 21 days after pollination stages were quantified by liquid chromatography tandem mass spectrometry (LC-MS/MS). The results from this work showed that glycolysis, oxidative pentose phosphate pathway (OPPP), tricarboxylic acid cycle and Ribulose 1,5-bisphosphate Carboxylase/Oxygenase (Rubisco) were active and contributed to FAS. In addition, glucose and glutamine were the most abundant sugar and amino acid, respectively, that embryos were using as carbon and nitrogen sources from the mother plant. Second, we have measured the efficiency with which pennycress embryos convert carbons from substrates into biomass (also known as carbon conversion efficiency, CCE). For this purpose, culture conditions mimicking the development of pennycress embryos in planta had to be developed. The major metabolites received by the embryo were identified by measuring the endosperm composition: hexoses, glutamine and abscisic acid. For developing culture conditions, embryos were incubated with varying concentrations of these substrates, polyethylene glycol (PEG) as well as light intensity for 6 days. Then, the culture conditions were validated when in vivo embryos had biomass accumulation rates similar to those from in planta embryos. The optimum culture condition contained glucose, glutamine, ABA, PEG and a light intensity of 20 mol/m2/s-1. Using these conditions, CCE of pennycress embryos was measured to be 93.4%. In addition, we have incubated embryos with i.) [U-13C6]-glucose and [1,2-13C2]-glucose, and ii.) [U-13C5]-glutamine for 6 days until isotopic steady-state was achieved. The labeling abundances in sugars, organic acids, phosphorylated compounds and organic acids were measured by LC-MS/MS using previously developed methods. In addition, we have developed a new high-throughput LC-MS/MS method that allows the analysis of the labeling in free AAs with a 11 min-run time, high accuracy and minimal matrix effects. From the labeling analyses by LC-MS/MS, there were main findings from 13C-glucose labeling. First, 60% of intracellular glucose was produced through the glucose 6-phosphate¿glucose cycle. Second, oxidative reactions of the PPP were more active in the cytosol than in the plastid. Lastly, aldolase was resynthesizing Fructose 1,6-bisphosphate. The 13C-glutamine experiment revealed that i.) gluconeogenesis was not significant; ii.) isocitrate dehydrogenase was reversible; iii.) plastidic NADP+-dependent malic enzyme was providing 17% of the carbon for de novo FAS; iv.) threonine aldolase was active; and v.) CO2 refixation by Rubisco contributed to 25% of 3-phosphoglycerate. The future work involves incorporating these findings into a mathematical model to generate a flux map with rates of biochemical reactions and a potential bottleneck in the metabolic network.