The release of small extracellular vesicles (sEV) has recently been reported, but knowledge of their function in neuron development remains limited. Using LC-MS/MS, we found that sEVs released from developing cortical neurons in vitro obtained from mice of both sexes were enriched in cytoplasm, exosome, and protein- and DNA/RNA-binding pathways. The latter included HDAC2, which was of particular interest because HDAC2 regulates spine development and populations of neurons expressing different levels of HDAC2 co-exist in vivo during the period of spine growth. Here, we found that HDAC2 levels decrease in neurons as they acquire synapses, and that sEVs from HDAC2-rich neurons regulate HDAC2 signaling in HDAC2-low neurons possibly through HDAC2 transfer. This regulation led to a transcriptional decrease in HDAC2 synaptic targets and the density of excitatory synapses. These data suggest that sEVs provide inductive cell-cell signaling that coordinates the development of dendritic spines via the activation of H...