Project Summary/Abstract Fundamental questions in developmental biology revolve around understanding the sequence of molecular and cellular events that regulates lineage specification and differentiation. Nowhere is this more poorly understood than in the early human embryo, which remains experimentally intractable for technical and ethical reasons past the onset of gastrulation - the point when the three major germ layers of the human body are specified within the embryonic epiblast tissue. Myself and others have previously developed stem cell-based systems that recapitulate key aspects of 3-dimensional (3D) mammalian development in vitro, paving the way to a more complete understanding of the specific steps that govern embryonic development. Here, I propose an innovative new experimental approach, using human pluripotent stem cells (hPSCs), combined with cutting-edge bioengineering technologies for a controllable, efficient and scalable modeling of human epiblast development in vitro. This model will permit studying the human germ layer differentiation trajectory and temporal dynamics in the correct 3D-conformation. I will combine state-of-the-art tools in genetics, imaging, and single-cell multi- omics in tandem with high-throughput computational analyses to define the key molecular and cellular events that regulate developmental patterning under different conditions. I will further investigate how these events are controlled by cellular metabolism and nutrient availability, which has important implications for understanding the early embryonic origin of numerous human diseases. My proposal will open up a completely new and powerful experimental paradigm to dissect the fundamental, inter-connected, principles of human developmental genetics at early embryonic stages that are otherwise inaccessible. This new knowledge will also directly inform efforts to efficiently generate mature tissues and cell types from stem cells for disease modeling and cell therapies. Ultimately, these findings will be critical for possible prevention of adverse pregnancy outcomes, offering a unique opportunity to understand the cellular and molecular mechanisms behind developmental disorders and congenital pathologies.