The function of the central nervous system (CNS) depends crucially upon the correct differentiation of neurons and formation of axonal connections. Some aspects of neuronal differentiation are known to occur as axonal connections are forming. Although serotonin is a highly conserved neurotransmitter that is important for many CNS functions, little is known about the process of serotonergic neuron differentiation. We show that in Drosophila, expression of the serotonin transporter (SerT) is both temporally and physically related to midline crossing. Additionally, we show that the axon guidance molecules roundabout2 and roundabout3 (robo2/3) are necessary for serotonergic neuron differentiation and function independently of their ligand, slit. Loss of robo2 or robo3 causes a loss of SerT expression in about half of neurons, and resembles the phenotype seen in mutants for the transcription factor eagle (eg). Finally, we show a direct relationship between robo2/3 and eg: robo2/3 mutants lose Eg expression in serotonergic neurons, and robo2 and eg interact genetically to regulate SerT expression. We propose that post-midline expression of Robo2/3 is part of a signal that regulates serotonergic neuron differentiation and is transduced by the transcription factor Eg.