Embryonic stem (ES) cells are pluripotent cells whose developmental state is equivalent to cells of the inner cell mass in the blastocyst-stage embryo. This unit presents a method of producing differentiated ES cells in which the cells are first aggregated on a less adhesive surface to form embryoid bodies (EBs). EBs are allowed to attach to a permissive substrate and then differentiate into neural cells in a serum-free medium. The resulting cell population can be maintained for several passages in serum-free medium in the presence of basic fibroblast growth factor (bFGF) or allowed to differentiate into postmitotic neurons by plating onto a glial monolayer followed by culture with serum. Also included in the unit are procedures for preparing feeder cells from embryonic fibroblasts and for preparing glial monolayer cultures, both for use in the main ES production protocol.