Brainstem median raphe (MR) neurons expressing the serotonergic regulator gene Pet1 send collateralized projections to forebrain regions to modulate affective, memory-related, and circadian behaviors. Some Pet1 neurons express a surprisingly incomplete battery of serotonin pathway genes, with somata lacking transcripts for tryptophan hydroxylase 2 ( Tph2 ) encoding the rate-limiting enzyme for serotonin [5-hydroxytryptamine (5-HT)] synthesis, but abundant for vesicular glutamate transporter type 3 ( Vglut3 ) encoding a synaptic vesicle-associated glutamate transporter. Genetic fate maps show these nonclassical, putatively glutamatergic Pet1 neurons in the MR arise embryonically from the same progenitor cell compartment—hindbrain rhombomere 2 (r2)—as serotonergic TPH2+ MR Pet1 neurons. Well established is the distribution of efferents en masse from r2-derived, Pet1 -neurons; unknown is the relationship between these efferent targets and the specific constituent source-neuron subgroups identified as r2- Pet1...