UV photolithography and hydrofluoric acid wet etching were used to produce silicon master molds and polydimethylsiloxane (PMDS)-based soft lithography was adopted to fabricate three-dimensional poly(lactic-co-glycolic acid) (PLGA) and PDMS microwell patterns with high aspect ratio and channel connection. Nine microwell patterns were thus obtained with different structural dimensions. Patterns were treated with oxygen plasma etching and polylysine coating to enhance hydrophilicity and cell compatibility for subsequent culture of C17. 2 neural stem cells. With proliferation during the culture, C17. 2 cells gradually distributed within the microwells, showing an obviously three-dimensional (3-D) growth behavior. The presence of channel structures greatly favored the 3-D growth of C17. 2 neural stem cells on the microwell patterns. Multi-layered scanning with confocal microscopy and 3-D rendering after carboxyfluorescein diacetate succinimidyl ester (CFDA-SE) staining showed that most C17. 2 cells grew within a range of 30 to 90 microm from the microwell bottom. Immunofluorescence staining indicated that C17. 2 cells within 3-D microwell patterns were uniformly nestin-positive on day 2 after cell plating. It could well be concluded that the microwell patterns thus fabricated were suitable for the 3-D culture and subsequent differentiation of C17. 2 neural stem cells. And the cells can be maintained with uniform stemness properties while cultured in these microwell patterns.