The proliferation and differentiation of endogenous neural stem cells and precursor cells in the postnatal central nervous system have been extensively reported. However, a full understanding on how to generate specific cell types to replace damaged or lost neurons and oligodendrocytes after spinal cord injury or other neurodegenerative disorders such as multiple sclerosis is still unclear. In the spinal cord in particular, there is a lack of understanding of how the use of exogenous compounds such as those that enhance endogenous acetylcholine signalling can effectively drive the proliferation and differentiation of endogenous stem cells (the ependymal cells) and oligodendrocyte precursor cells. Using the thymidine analogue 5-ethynyl-2-deoxyuridine (EdU) as a marker of proliferating cells, we show that adult C57/Bl6 mice administered donepezil (i.p 5µM) – an acetylcholinesterase inhibitor and the α7 nicotinic acetylcholine receptor (nAChR) positive allosteric modulator (PNU-120596, 1µM) had significantly (P < 0.0001) more EdU positive cells, compared to control, in the central canal, grey and white matter of uninjured spinal cord. Furthermore, in these animals, there was a significantly higher (P < 0.0001) number of EdU+ cells that also expressed the oligodendrocyte marker, PanQKI in both the grey and white matter compared to control. There was also a significantly (P < 0.0001) higher extent of colocalisation of EdU with the neuronal marker HUC/D in the spinal cord grey matter of animals treated with donepezil in combination with PNU-120596 compared to control animals. In a lysolecithin-induced focal demyelination mouse model, treatment with either PNU-120596 alone or in combination with donepezil significantly enhanced cell proliferation and oligodendroglial differentiation compared to lysolecithin alone treated animals. Thoraco-lumbar spinal cord slices cultured in the presence of donepezil and the muscarinic receptor inhibitor atropine revealed that most of the cholinergic proliferative response is mediated via nicotinic acetylcholine receptors in the central canal, grey and white matter; there was a significantly higher number of EdU positive cells in donepezil + atropine treated slices compared to donepezil alone. Slices cultured in the presence of donepezil, atropine and the non- α7nAChR inhibitor DHbE had a significantly higher number of EdU positive cells compared to slices cultured with donepezil alone, donepezil + atropine or the nicotinic agonist cytisine + DHbE in the central canal of slices; suggesting that cell proliferation is mostly mediated through α7nAChRs. Finally, the calcineurin-nuclear factor of activated T cells (NFAT) calcium signalling pathway downstream of α7nAChRs plays a role in cells proliferation in vitro.