Altered expression of the human FEV (fifth Ewing variant) ETS transcription factor gene impacts the level of CNS serotonin (5-HT) neuron gene expression and maternal nurturing. However, the regulatory mechanisms that determine FEV expression are poorly understood. Here, we investigated the cis-regulatory control of FEV to begin to identify the upstream transcription factors that restrict FEV expression to 5-HT neurons. We find that sequences extending only 275 bp upstream of the FEV 5' untranslated region are sufficient to direct FEV transgene expression to embryonic 5-HT neurons, although sequences farther upstream are required for maintenance in adult 5-HT neurons. Two highly conserved consensus GATA factor binding sites within the 275 bp region interact with GATA factors in vitro. Chromatin immunoprecipitations with embryonic hindbrain demonstrated Gata-2 interactions with the orthologous mouse Pet-1 ETS cis-regulatory region. Mutagenesis of GATA sites revealed that one or the other site is required for serotonergic FEV transgene expression. Unexpectedly, FEV-LacZ transgenes enabled determination of 5-HT neuron precursor fate in the adult Pet-1(-/-) dorsal and median raphe nuclei and thus provided additional insight into FEV/Pet-1 function. Comparable numbers of FEV-LacZ-positive cells were detected in Pet-1(+/-) and Pet-1(-/-) adult dorsal raphe nuclei, indicating that the majority of mutant serotonergic precursors are not fated to apoptosis. However, B7 dorsal raphe cells were aberrantly distributed, suggesting a role for FEV/Pet-1 in their midline organization. Our findings identify a direct transcriptional interaction between Gata-2 and FEV and a unique marker for new insight into FEV/Pet-1 function in 5-HT neuron development.