A detailed protocol is described allowing the generation of essentially pure populations of glutamatergic neurons from mouse embryonic stem (ES) cells. It is based on the culture of ES cells that are kept undifferentiated by repeated splitting and subsequently amplified as non-adherent cell aggregates. Treatment with retinoic acid causes these ES cells to essentially become neural progenitors with the characteristics of Pax6-positive radial glial cells. As they do in vivo, these progenitors differentiate in glutamatergic pyramidal neurons that form functional synaptic contacts and can be kept in culture for long periods of time. This protocol does not require the use of ES lines expressing resistance or fluorescent markers and can thus be applied in principle to any wild-type or mutant ES line of interest. At least 2 weeks are required from starting ES cell culture until plating progenitors and differentiating neurons establish synaptic transmission within about 10 days.