Abstract A critical process for early development of the vertebrate embryo is induction of the three primary germ layers. Knowledge of the molecules that are produced in each embryonic cell is essential to understanding their function to pattern the embryonic body. Decades of innovative cell biological and embryological studies, assessment of function one gene at a time, and recently deep transcriptomics profiling via next-generation sequencing have exposed the developmental roles of important genes, transcripts, and some proteins. As a result, scientists have defined the spatial and temporal changes of mRNAs and abundant proteins, and some serendipitously identified metabolites of importance, such as folate assisting in closure of the neural tube. However, it has been technologically impossible to utilize high-resolution mass spectrometry (HRMS), the gold standard technology for proteomics and metabolomics (`omics), to study hundreds of metabolites and thousands proteins in single embryonic cells in the vertebrate embryo. Further, in developing systems, a complex correlation between gene transcription and translation as well as posttranslational modifications complicate the use of mRNA information to approximate protein and metabolite levels. Without the availability of single-cell HRMS as a routine laboratory tool or other technologies capable of deep metabolomics and proteomics, scientists at present lack insights into metabolic processes that contribute to early embryonic patterning. The research program proposed here fills this enormous knowledge and technological gap by utilizing ultrahigh-sensitivity HRMS platforms that were custom-designed, custom-built, and validated in the vertebrate frog (Xenopus laevis) embryo, a popular model in cell/developmental biology, to understand noncanonical metabolomic processes that control formation of the germ layers. Most recently, single-cell HRMS in X. laevis has discovered metabolites that are capable of (i) altering the normal cell fates of embryonic cells, (ii) communicating between blastomeres, and (iii) affecting the whole-organismal performance of the resulting tadpole. These findings have shed light on a gap in our basic knowledge of molecules participating in the successful induction of the germ layers. This research program will determine roles that metabolites play in patterning the embryonic body plan. This work will integrate classical embryological manipulations, cell-fate tracking, and fluorescent microscopy with new-generation quantitative mass spectrometry capable of subcellular sensitivity to characterize how targeted metabolic reactions impact reproducible tissue fates in the X. laevis embryo. Because these molecular processes are highly conserved across vertebrates, the data collected from Xenopus are likely to have high relevance to human development. This interdisciplinary research program will help discover metabolic mechanisms governing cell differentiation and embryogenesis.