Multipotent neural stem cells (NSCs) are widely applied in pre-clinical and clinical trials as a cell source to promote tissue regeneration in neurodegenerative diseases. Frequently delivered as dissociated cells, aggregates or self-organized rosettes, it is unknown whether disruption of the NSC rosette morphology or method of formation affect signaling profiles of these cells that may impact uniformity of outcomes in cell therapies. Here we generate a neural cell–cell interaction microchip (NCCIM) as an in vitro platform to simultaneously track an informed panel of cytokines and co-evaluate cell morphology and biomarker expression coupled to a sandwich ELISA platform. We apply multiplex in situ tagging technology (MIST) to evaluate ten cytokines (PDGF-AA, GDNF, BDNF, IGF-1, FGF-2, IL-6, BMP-4, CNTF, β-NGF, NT-3) on microchips for EB-derived rosettes, single cell dissociated rosettes and reformed rosette neurospheres. Of the cytokines evaluated, EB-derived rosettes secrete PDGF-AA, GDNF and FGF-2 prominently, whereas this profile is temporarily lost upon dissociation to single cells and in reformed neurospheres two additional cytokines, BDNF and β-NGF, are also secreted. This study on NSC rosettes demonstrates the development, versatility and utility of the NCCIM as a sensitive multiplex detector of cytokine signaling in a high throughput and controlled microenvironment. The NCCIM is expected to provide important new information to refine cell source choices in therapies as well as to support development of informative 2D or 3D in vitro models including areas of neurodegeneration or neuroplasticity.