Isolated neuroblasts from gastrula-stage Drosophila embryos divide and differentiate in vitro to produce clonally derived clusters of neurons. Both serotonin and dopamine are expressed within these cultures in patterns that are similar to their distributions in vivo. Clusters containing serotonergic neurons are generally distinct from those with dopaminergic neurons, suggesting that different neuroblasts produce neurons with these phenotypes. The appearance of each transmitter correlates with transcription of dopa decarboxylase in the transmitter-positive cells. The developmental program leading to the appearance of either serotonergic or dopaminergic neurons is different for each transmitter type. Thus, serotonergic cells are progeny of early neuroblast divisions, whereas dopaminergics arise throughout the lineage. Inhibition of cell division, but not nuclear division, with cytochalasin B demonstrates that the expression of the serotonin phenotype requires a determined number of DNA replications. These experiments establish that neuroblasts, as soon as they are formed during early gastrulation events in Drosophila, are already determined for the subsequent expression of transmitter phenotype.