How a common neurotransmitter phenotype specified in neurons of different origins is an outstanding issue in neuronal development and function. In C. elegans larvae, serotonin is synthesized in 2 pairs of neurons, the secretory neurons NSM and the chemosensory neurons ADF. In order to delineate the molecular mechanisms of serotonergic phenotype establishment, we have screened for neuron-specific serotonin deficient (nss) mutants. Our prior study showed that the POU-homeodomain factor UNC-86 is expressed in and required for the NSM neurons to adopt serotonergic phenotype and correct pathfinding, whereas ADF are unaffected in unc-86-null mutants. Here, we report that the LIM-homeodomain factor LIM-4 regulates ADF serotonergic phenotype. In lim-4 mutants, many aspects of ADF differentiation occur, however, they fail to express serotonin phenotype and exhibit aberrant cilia properties. LIM-4 expression rises in the neuroblast that produces two distinct neurons: ADF and the olfactory neuron AWB. We show that lim-4 is regulated by separable mechanisms to determine disparate subtype identities in these two neuronal types. In vivo promoter analyses reveal that cis-element(s) within introns are necessary and sufficient to direct lim-4 to specify serotonergic phenotype, whereas its 5'-upstream sequence directs lim-4 function in AWB. Thus, a transcription factor may act independently to specify distinct differentiation traits in two sister cells. We propose that serotonergic identity is specified in cell-specific contexts to coordinate the development and function.