Statement of Purpose: The body’s complement of blood and immune cells is produced by a small population of hematopoietic stem cells (HSCs). The complex process of hematopoiesis is governed by the activation and quiescence of HSCs within the bone marrow niche. Within the niche, physical, chemical, and cellular cues combine in spatially and temporally defined zones to elicit responses from the residing HSCs. Synthetic culture systems for HSCs have been hindered by the synergistic coupling of these factors. Specifically, biomaterial properties impact long-range cell-cell signaling through modulation of soluble factors. Previous work within the lab has characterized HSC differentiation within co-culture platforms of mesenchymal stromal cells (MSCs), in methacrylamide-functionalized gelatin (GelMA) hydrogels. Here we explore how the biomaterial impacts differentiation patterns through the secondary effect of secreted factors by co-cultured niche cells. By combining biomaterial design and secretome analysis, we seek to leverage initial matrix properties to direct HSCs through both mechanical properties and modulation of biotransport of cell-secreted factors. Herein I describe the analysis of soluble factors from an HSC biomaterial culture. An iterative process was used to establish a Partial Least Squares (PLS) model with a minimum cocktail of cytokines that correlated to an increase in the HSC population. Through secretome analysis of HSCs and MSCs within mechanically defined gelatin hydrogel platforms, we demonstrate a crosstalk between biomaterial design and cell-secreted factors, and the synergistic impact on stem cell differentiation patterns.