During the development of the nervous system, the acquisition of the GABA neurotransmitter phenotype is crucial for neural networks operation. Although both intrinsic and extrinsic signals such as transcription factors and growth factors have been demonstrated to govern the acquisition of GABA, few data are available concerning the effects of modulatory transmitters expressed by axons that progressively invade emerging neuronal networks. Among such transmitters, serotonin (5-HT) is a good candidate because serotonergic axons innervate the entire CNS at very early stages of development. We have shown previously that descending 5-HT slows the maturation of inhibitory synaptic transmission in the embryonic mouse spinal cord. We now report that 5-HT also regulates the spatiotemporal changes of the GABAergic neuronal population in the mouse spinal cord. Using a quantitative confocal study performed on acute and cultured spinal cords, we find that the GABAergic population matures according to a similar rostrocaudal temporal gradient both in utero and in organotypic culture. Moreover, we show that 5-HT delays the appearance of the spinal GABAergic system. Indeed, in the absence of 5-HT descending inputs or exogenous 5-HT, the GABAergic population matures earlier. In the presence of exogenous 5-HT, the GABA population matures later. Finally, using a pharmacological approach, we show that 5-HT exerts its action via the 5-HT1 receptor family. Together, our data suggest that, during the course of the embryonic development, 5-HT descending inputs delay the maturation of lumbar spinal motor networks relative to brachial networks.