Electrical activation of hippocampal neurons can cause calcium influx through different entry sites which may specify nuclear signalling and induction of gene transcription and downstream physiological outputs. Genomic responses initiated by NMDA receptors (NMDARs) are critically dependent on whether synaptically or extrasynaptically located receptors are stimulated; calcium flux through synaptic NMDARs activates CREB whereas flux through extrasynaptic NMDARs triggers a CREB shut-off signal. Here we investigated the possibility that the coupling of extrasynaptic NMDARs to the CREB shut-off pathway is regulated during in vitro development. Cultured hippocampal neurons were analyzed after 7 or 12 days of in vitro culturing. We found that synaptic NMDAR activity induced CREB phosphorylation at day in vitro (DIV) 7 and DIV 12. In contrast, the extrasynaptic NMDAR-dependent CREB shut-off signal is developmentally regulated. At DIV 12 extrasynaptic NMDAR activation shuts down CREB and overrides the CREB-activating signal triggered by synaptic NMDAR activation. In contrast, at DIV 7 this shut off signal is absent; both synaptic and extrasynaptic NMDARs activate CREB function. Developmental changes in NMDAR signaling have been proposed to contribute to the emergence of glutamate excitotoxicity, which causes apoptosis or necrosis depending on the severity of the insult. Since CREB regulates a number of pro-survival genes, the emergence of this shut-off around DIV 7 may contribute to the increase in susceptibility of neurons to glutamate-induced neuropathology in vitro and in vivo during post-natal development.