Growing and differentiating human stem cells in vitro can provide access to study the molecular mechanisms that control cellular development in a manner pertinent to human embryogenesis. To fully understand such processes, however, it is important to recreate culture conditions that most closely relate to those in living tissues. As step in this direction, we have developed a robust three-dimensional cell culture system using inert highly porous solid matrices manufactured from polystyrene that can be routinely used to study the differentiation of human pluripotent stem cell-derived neurons in vitro. Neurite outgrowth was significantly enhanced when neurons were grown in a three-dimensional environment compared to traditional flat surfaces and resulted in the formation of extensive neural networks. These data suggest that the topography within the culture environment can significantly alter cell development and will therefore be an important feature when investigating the potential of human stem cells. (C) 2003 Elsevier Inc. All rights reserved.