Recently, d-serine has been identified as an important NMDA-receptor co-agonist, which might play a role in central nervous system development. We investigated this by studying rat P19 cells, an established model for neuronal and glial differentiation. Our results show that (1) the d-serine synthesizing enzyme serine racemase was expressed upon differentiation, (2) extracellular d-serine concentrations increased upon differentiation, which was inhibited by serine racemase antagonism, and (3) inhibition of d-serine synthesis or prevention of d-serine binding to the NMDA-receptor increased synaptophysin expression and intercellular connections, supporting a role for NMDA-receptor activation by d-serine, synthesized by serine racemase, in shaping synaptogenesis and neuronal circuitry during central nervous system development. In conjunction with recent evidence from literature, we therefore suggest that d-serine deficiency might be responsible for the severe neurological phenotype seen in patients with serine deficiency disorders. In addition, this may provide a pathophysiological mechanism for a role of d-serine deficiency in psychiatric disorders.