Three-dimensional (3-D) spheroids are widely used for culturing cells. However, 2-dimensional (2-D) monolayer cultures have also been adopted for culture and used in a broad range of cell biology studies. To address the effect of dimensionality on the growth and differentiation of neuroprogenitor cells in 3-D spheroids and 2-D monolayer cultures, cells were isolated from cerebral cortex, cerebella and brainstem of fetal rat brain then cultured in serum-free DMEM/F12 medium or DMEM with 10% FBS. The growth and differentiation of neuroprogenitor cells from three brain regions in spheroids was compared with that in monolayer cultures, and the differentiation components of neuroprogenitor cells were compared with in vivo brain sections. Neuroprogenitor cells in spheroids proliferate actively over 10 days in culture as showed by Ki67 incorporation and increase in spheroid diameter. More neuroprogenitor cells underwent neuronal differentiation in spheroids than in monolayer cultures. In comparison with fixed rat brain sections, the neuron to astrocyte ratio, as shown by neurofilament to glial fibrillary acidic protein immunoreactivity, in spheroids is similar to that found in adult rat tissue sections. Our results suggest that the spheroid culture system mimics the in vivo cytoarchitecture to a greater extent and more closely reflects the cellular composition in adult brain tissue. This supports the notion that the intercellular niche in spheroids is more favorable for the survival and differentiation of neuronal precursors, while the cues in monolayer cultures may favor glial cell survival. It is therefore concluded that dimensionality plays a significant role in determining cellular behavior in vitro.