Astrocytes release numerous factors known to contribute to the process of synaptogenesis, yet knowledge about the signals that control their release is limited. We hypothesized that neuron-derived signals stimulate astrocytes, which respond by signaling back to neurons through the modulation of astrocyte-released synaptogenic factors. Here we investigate the effect of cholinergic stimulation of astrocytes on synaptogenesis in co-cultured neurons. Using a culture system where primary rat astrocytes and primary rat neurons are first grown separately allowed us to independently manipulate astrocyte cholinergic signaling. Subsequent co-culture of pre-stimulated astrocytes with naïve neurons enabled us to assess how prior stimulation of astrocyte acetylcholine receptors uniquely modulates neuronal synapse formation. Pre-treatment of astrocytes with the acetylcholine receptor agonist carbachol increased the expression of synaptic proteins, the number of pre- and postsynaptic puncta, and the number of functional synapses in hippocampal neurons after 24 hours in co-culture. Astrocyte secretion of the synaptogenic protein thrombospondin-1 increased after cholinergic stimulation and the inhibition of the target receptor for thrombospondins prevented the observed increase in neuronal synaptic structures. Thus, we identified a novel mechanism of neuron-astrocyte-neuron communication, i.e. , neuronal release of acetylcholine stimulates astrocytes to release synaptogenic proteins leading to increased synaptogenesis in neurons. This study provides new insights into the role of neurotransmitter receptors in developing astrocytes and into our understanding of the modulation of astrocyte-induced synaptogenesis.