Stem cell research has been greatly facilitated by comprehensive and integrative multi-omics studies. As a unique approach of functional analysis, metabolomics measures many metabolites and activities of metabolic pathways which can directly indicate cellular energetic status, cell proliferation and fitness, and stem cell fate choices such as self-renewal versus differentiation. Here we describe the methods of applying metabolomics,
C-labeled glucose and glutamine tracing with mouse embryonic stem cells (ES cells), metabolite analysis using mass spectrometry tools, and the following statistical and computational modeling analysis. Integration of these methods into the more common gene expression and epigenetics analysis toolbox will help to generate a more complete picture and in-depth understanding of one's stem cells of interest.