This chapter focuses on three-dimensional (3D) models prepared from human stem- or neural precursor cells, and on aggregating rat brain cell cultures. Patches of polytetrafluoroethylene membranes bearing the 3D neural tissues were placed upside down onto multi-electrode array systems. The level of maturation of the TH-positive neurons was higher in 3D cultures, where many varicose neurites could be found. The 3D rat brain cell model first developed by Moscona and coworkers has been widely characterized and used for neurotoxicological and Developmental Neurotoxicity (DNT) studies by Honegger and co-workers. A good in vitro model for DNT must replicate the critical developmental steps such as cell proliferation, migration, process formation, as well as synapse formation and myelination. The importance of a heterogenic cell culture model for DNT is further supported by the fact that both neurons and glial cells can be the primary targets of neurotoxic insults.