In early embryonic development, spatial gradients of diffusible signaling molecules play important roles in controlling differentiation of cell types or arrays in diverse tissues. Thus, the concentration of exogenous cytokines or growth factors at any given time is crucial to the formation of an enriched population of a desired cell type from primitive stem cells in vitro. Microfluidic technology has proven very useful in the creation of cell-friendly microenvironments. Such techniques are, however, currently limited to a few cell types. Improved versatility is required if these systems are to become practically applicable to stem cells showing various plasticity ranges. Here, we built a microfluidic platform in which cells can be exposed to stable concentration gradients of various signaling molecules for more than a week with only minimal handling and no external power source. To maintain stability of the gradient concentration, the osmotic pumping performance was optimized by balancing the capillary action and hydraulic pressure in the inlet reagent reservoirs. We cultured an enriched population of neural progenitors derived from human embryonic stem cells in our microfluidic chamber for 8 days under continuous cytokine gradients (sonic hedgehog, fibroblast growth factor 8, and bone morphogenetic protein 4). Neural progenitors successfully differentiated into neurons, generating a complex neural network. The average numbers of both neuronal cell body clusters and neurite bundles were directly proportional to sonic hedgehog concentrations in the gradient chip. The system was shown to be useful for both basic and translational research, with straightforward mechanisms and operational schemes.