Morphogen gradients play a critical role in directing cell fate specification throughout development but are not yet implemented by current in vitro models. Human induced pluripotent stem cell derived brain organoids recapitulate early cellular and molecular mechanisms of brain development. Because they lack spatial organization to fully mimic the in vivo topographical organization, we introduce a new method to model early neural tube patterning by exposing organoids to continuous gradients within a five-chamber mesofluidic device. We hypothesized that the introduction of oriented signaling centers over a continuum culture system of brain organoids would induce regional specification in a distance-dependent manner. Our gradient patterning device has the advantage of not relying on externally driven flow, but only on short-range diffusion, simplifying the approach and offering a wide range of applications. We demonstrate by scRNA-seq that this “brain-on-a-chip” platform serves to spatially control cellular fate decisions. The device induces antero-posterior and dorsal-ventral patterning, mirroring gene expression levels of transcription factors specific of different human brain regions. This practical and versatile device generates multiple neuronal lineages from cortex, striatum, thalamus and cerebellum and will facilitate continued research on signaling cascades and pathways that underlie neural tube patterning and tissue organization, offering insights into interindividual variability in neurotypical and patient-derived iPSC lines.