Production of dopaminergic (DA) neurons from stem/precursor cells for transplantation in Parkinson's disease has become a major focus of research. However, the inductive signals mediating the production of DA neurons remain poorly understood, and the influence of other cell populations simultaneously generated within the cell aggregates has not been studied. We investigated whether DA phenotype (i.e. tyrosine hydroxylase-immunoreactive, TH-ir), serotonergic, floor plate (FP4-ir), and fibroblast growth factor 8 (FGF-8)-ir cells differentiate from proliferating cell aggregates obtained from rat mesencephalic precursors, and we also investigated the effects of serotonergic cells on differentiation of DA cells. We observed FP4-ir, FGF-8-ir, TH-ir and serotonergic cells within the aggregates. The TH-ir cells appeared within or in close proximity to a central FP4-ir core, and then concentrated peripherally forming a cap that surrounded the central FP4-ir area. The serotonergic cells and fibers formed a cap surrounding that of TH-ir neurons. Cell aggregates treated with an antibody against FGF-4 or with the serotonergic toxin 5,7-dyhydroxytryptamine or the serotonin synthesis inhibitor dl-p-chlorophenylalanine showed a marked decrease in the number of 5-HT-ir cells (10–20% of controls) and a marked increase in that of TH-ir neurons (700–900% of controls). The present results show that manipulation of other cell populations in the cell aggregates, particularly the serotonergic population, may be an effective method of increasing the production of DA neurons from stem/precursor cells.