Background: Reproduction in animals requires development of distinct neurons in each sex. In C. elegans, most ventral cord neurons (VCNs) are present in both sexes, with the exception of six hermaphrodite-specific neurons (VCs) and nine pairs of male-specific neurons (CAs and CPs) that arise from analogous precursor cells. How are the activities of sexual regulators and mediators of neuronal survival, division, and fate coordinated to generate sex-specificity in VCNs? Results: To address this, we have developed a toolkit of VCN markers that allows us to examine sex-specific neurogenesis, asymmetric fates of daughters of a neuroblast division, and regional specification on the anteroposterior axis. Here, we describe the roles of the Hox transcription factors LIN-39 and MAB-5 in promoting survival, differentiation, and regionalization of VCNs. We also find that the TALE class homeodomain proteins CEH-20 and UNC-62 contribute to specification of neurotransmitter fate in males. Furthermore, we identify that VCN sex is determined during the L1 larval stage. Conclusions: These findings, combined with future analyses made possible by the suite of VCN markers described here, will elucidate how Hox-mediated cell fate decisions and sex determination intersect to influence development of neuronal sex differences. Developmental Dynamics 243:159–171, 2014. © 2013 Wiley Periodicals, Inc.