Replicating the cascade of signals necessary to control stem cell behavior remains a central challenge for the regenerative medicine community. The hematopoietic system offers an ideal biological system to motivate the development of innovative technologies needed to accomplish this goal. Hematopoiesis is the process where the body’s blood and immune cells are generated from a small number of hematopoietic stem cells (HSCs). HSC quiescence, self-renewal, and differentiation take place in, and are regulated by, unique regions of the bone marrow termed niches. Many innovations in stem cell engineering first focused on replicating constellations of extracellular matrix, biomolecular, or metabolic (e.g., hypoxia) signals within the niche. For example, we developed microfluidic approaches to create gelatin hydrogels containing marrow-inspired gradients of stiffness, niche cells, and biomolecules for extended culture of 103-104 primary murine HSCs. However, signaling between cohorts of different cell populations within the niche is also a critical regulator of stem cell expansion, quiescence, and lineage specification and may contribute to hematopoietic cancers. We adapted our platform to show the kinetics of HSC-niche cell crosstalk can be manipulated via hydrogel network parameters to dramatically alter HSC fate. We also developed bioinformatics tools to identify secretome signals generated by marrow mesenchymal stem cells (MSCs) that enhance retention of quiescent HSCs. However, tools to study reciprocal signaling between multiple cell populations within an engineered stem cell niche remains limited by our ability to locally control the assembly, culture, and recovery of multicellular cohorts. Conventional bulk hydrogels do not allow an avenue to tailor, or trace the evolution of, the local microenvironment surrounding unique cell subpopulations. Our research community requires a new tissue engineering ecosystem that allows us to replicate dynamic, multicellular stem cell niches and also exploit recent advances in single-cell sequencing and bioinformatics. The primary objective of this NIDDK Catalytic Tool and Technology Development project (R21 DK131751-01) is to develop underlying technology required to form a granular stem cell niche. Granular hydrogels are macroscale structures generated as jammed assemblies of microscale hydrogel particles. To date they have been predominantly used as acellular hydrogel particles with cells cultured in the voids between particles. Our innovative approach will encapsulate single marrow derived hematopoietic cells in distinct nanoliter-volume hydrogel microdroplets that can be rapidly formed, tailored for each discrete cell population, and non-toxically degraded. We will use the short diffusion lengths of microdroplets to study of the convergence of matrix biophysical and metabolic signals on HSC fate. We address the high-risk, high-reward nature of this catalytic tool development project via the following aims: ...