Stem cell self-renewal implies proliferation under continued maintenance of multipotency. Small changes in stem cell numbers may lead to large differences in differentiated cell numbers, resulting in significant physiological consequences. Proliferation is typically regulated in the G1-phase which is associated with differentiation and cell cycle arrest. However, embryonic stem (ES) cells may lack a G1- phase checkpoint2. Regulation of proliferation in the DNA damage S/G2-phase cell cycle checkpoint pathway is known for its role in maintenance of the chromatin structural integrity. We provide evidence that an auto/paracrine g-amino butyric acid (GABA) signaling via GABAA receptors negatively controls embryonic stem (ES) cell, peripheral neural crest stem (NCS) cell proliferation, preimplantation embryonic growth as well as proliferation in the adult brain stem cell niche. GABAA receptor activation leads to hyperpolarization, increased cell volume and accumulation of stem cells in the cell cycle S- phase, thereby causing a rapid reduction in cell proliferation. GABAA receptors signal through S- phase checkpoint kinases of the PI3K related kinase (PIKK) family and histone variant H2AX. This signaling pathway critically regulates proliferation independently of differentiation, apoptosis and overt DNA damage. Our results suggest a fundamentally different mechanism of proliferation control in these stem cells, compared to most somatic cells, involving proteins in the DNA damage checkpoint pathway.