<p>The functional complexity and anatomical organization of the nervous system are established during regional patterning of its embryonic precursor—the neural tube. Human pluripotent stem (hPS) cell-based models have emerged as valuable complements to animal models for studying neural development. Here we present the design and implementation of a microfluidic gradient device for modeling human neural tube formation and regional patterning with hPS cells. The microfluidic device enables the formation of tubular or spherical colonies of hPS cells at prescribed locations within microfluidic channels, allowing the cell colonies to form lumenal structures while being exposed to well-controlled chemical gradients for rostral–caudal and/or dorsal–ventral patterning, resulting in the formation of a microfluidic neural tube-like structure (μNTLS) or a forebrain-like structure (μFBLS). The μNTLS recapitulates important hallmarks of early human neural development, including well-defined lumenal morphologies, spatially organized regional marker expression, emergence of secondary signaling centers and the development of neural crest cells. The dorsal–ventral patterned μFBLS further recapitulates spatially segregated dorsal and ventral regions, as well as the layered segregation of early neurons from neural progenitors, mimicking human forebrain pallium and subpallium development. Both the μNTLS and μFBLS are compatible with long-term culture, live imaging, immunofluorescence staining and single-cell sequencing, serving as robust systems for studying human neurodevelopment and disease. This protocol can be implemented by a researcher with polydimethylsiloxane soft lithography and cell culture experience and takes ~8–41 d to complete, depending on the types of neural structure to model and their developmental stages, with an option for prolonged culture to promote neuronal maturation.</p>