Spatial heterogeneity plays a key role in the development and function of human tissues and therefore needs to be incorporated within in vitro models to maximise physiological relevance and predictive power. Here, we developed and optimised methods to generate spatial heterogeneity of hydrogel-embedded bioactive signalling molecules within organ-on-a-chip (OOAC) systems, to drive spatiotemporal tissue patterning through controlled stem cell differentiation. As an exemplar application, we spatially patterned bone morphogenetic protein-2 (BMP-2) in both closed-channel and open-chamber OOAC formats. The resulting BMP-2 gradient in 3D heparin methacryloyl/gelatin methacryloyl, successfully drove spatially divergent differentiation of human bone marrow-derived stem cells into bone-like and cartilage-like regions, mimicking the process of endochondral ossification in the growth plate. The application of hydrogel-embedded morphogens to drive spatial tissue patterning within OOAC systems represents a significant technological advancement and has broad-ranging applicability for a diverse range of tissues and organs, and a wide variety of OOAC platforms.