The bone marrow provides spatially and temporally variable signals that impact the behavior of hematopoietic stem cells (HSCs). While multiple biomolecular signals and bone marrow cell populations have been proposed as key regulators of HSC fate, new tools are required to probe their importance and mechanisms of action. Here, a novel method based on a microfluidic mixing platform to create small volume, 3D hydrogel constructs containing overlapping patterns of cell and matrix constituents inspired by the HSC niche is described. This approach is used to generate hydrogels containing opposing gradients of fluorescent microspheres, MC3T3-E1 osteoblasts, primary murine hematopoietic stem and progenitor cells (HSPCs), and combinations thereof in a manner independent of hydrogel density and cell/particle size. Three different analytical methods are described to characterize local properties of these hydrogels at multiple scales: 1) whole construct fluorescent analysis; 2) multi-photon imaging of individual cells within the construct; 3) retrieval of discrete sub-regions from the hydrogel post-culture. The approach reported here allows the creation of stable gradients of cell and material cues within a single, optically translucent 3D biomaterial to enable a range of investigations regarding how microenvironmental signals impact cell fate.