<p>Emerging evidence suggests that individual neurons can utilize multiple neurotransmitters, challenging traditional views of neuronal identity. However, as most studies to date have focused on the adult stage, a fundamental question remains: does this dual-transmitter phenotype emerge only in adulthood, or does it have specific developmental origins? To address this, we performed a comprehensive whole-brain screen across various postnatal stages (P0–P60) in mice. Using a dual Flp/Cre-mediated intersectional labeling strategy to drive fluorescent protein expression, we genetically marked glutamatergic (Vglut2⁺) and GABAergic (Vgat⁺) neurons. Our results reveal that dual-transmitter neurons are not exclusive to the adult brain but are already prevalent during development. Notably, the emergence of these neurons is asynchronous across brain regions, with some appearing at P0 and others gradually emerging at later time points. Furthermore, direct mRNA in situ detection confirmed the concomitant expression of both markers in selected regions, establishing that these dual-transmitter neurons arise through co-expression rather than a developmental switch between neurotransmitters, at least within the brain regions we examined. Overall, our study deepens the understanding of dual-transmitter phenomena by demonstrating that this phenotype is established during development and follows region-specific temporal trajectories.</p>