Abstract.Maturation of functional neuronal circuits during central nervous system development relies on sophisticated mechanisms. First, axonal and dendritic growth should reach appropriate targets for correct synapse elaboration. Second, pruning and neuronal death are required to eliminate redundant or inappropriate neuronal connections. Serotonin, in addition to its role as a neurotransmitter, actively participates in postnatal establishment and refinement of brain wiring in mammals. Brain resident macrophages, i.e. microglia, also play an important role in developmentally-regulated neuronal death as well as in synaptic maturation and elimination. Here, we tested the hypothesis of cross-regulation between microglia and serotonin during postnatal brain development in a mouse model of synaptic refinement. We found expression of the serotonin 5-HT 2B receptor on postnatal microglia, suggesting that serotonin could participate in temporal and spatial synchronization of microglial functions. Using two-photon microscopy, acute brain slices and local delivery of serotonin, we observed that microglial processes moved rapidly toward the source of serotonin in Htr 2B +/+ mice, but not in Htr 2B-/-mice lacking the 5-HT 2B receptor. We then investigated whether some developmental steps known to be controlled by serotonin, could potentially result from microglia sensitivity to serotonin. Using an in vivo model of synaptic refinement during early brain development, we investigated the maturation of the retinal projections to the thalamus and observed that Htr 2B -/-mice present anatomical alterations of the ipsilateral projecting area of retinal axons into the thalamus. In addition, activation markers were upregulated in microglia from Htr 2B -/-compared to control neonates, in the absence of apparent morphological modifications. These results support the hypothesis that serotonin interacts with microglial cells and these interactions participate in brain maturation.