Gastrulation is a critical stage in embryonic development during which the germ layers are established. The advances and increasing availability of sequencing technologies led to the identification of gene regulatory programs that control the emergence of the germ layers and its derivatives. However, proteome-based studies of early mammalian development are scarce. To overcome this, we utilized gastruloids and a multilayered mass spectrometry-based proteomics approach to investigate the global dynamics of (phospho)protein expression during gastruloid differentiation. Our findings revealed that many proteins exhibit temporal expression with unique expression profiles corresponding to the three germ layers, which we also validated using single-cell proteomics technology. Notably, detected temporal protein expression dynamics in gastruloids aligns with equivalent mouse embryo stages. Additionally, we profiled enhancer interaction landscapes using P300 proximity labeling, which revealed numerous gastruloid-specific transcription factors and chromatin remodelers. Subsequent degron-based perturbations combined with scRNA-seq identified a critical role for ZEB2 in regulating mouse and human somitogenesis. Overall, this study provides a rich resource for developmental and synthetic biology communities endeavoring to understand mammalian embryogenesis.