Cell culture systems are widely used for molecular, genetic and biochemical studies. Primary cell cultures of animal tissues offer the advantage that specific cell types can be studied in vitro outside of their normal environment. We provide a detailed protocol for generating primary neural cell cultures derived from larval brains of Drosophila melanogaster. The developing larval brain contains stem cells such as neural precursors and intermediate neural progenitors, as well as fully differentiated and functional neurons and glia cells. We describe how to analyze these cell types in vitro by immunofluorescent staining and scanning confocal microscopy. Cell type-specific fluorescent reporter lines and genetically encoded calcium sensors allow the monitoring of developmental, cellular processes and neuronal activity in living cells in vitro. The protocol provides a basis for functional studies of wild-type or genetically manipulated primary neural cells in culture, both in fixed and living samples. The entire procedure takes ∼3 weeks.