ES (embryonic stem) cell lines are derived from the epiblast of pre-implantation embryos and like the inner cell mass cells from which they are derived exhibit the remarkable property of pluripotency, namely the ability to differentiate into all cell lineages comprising the adult organism. ES cells and their differentiated progeny offer tremendous potential to regenerative medicine, particularly as cellular therapies for the treatment of a wide variety of chronic disorders, such as Type 1 diabetes, Parkinson's disease and retinal degeneration. In order for this potential to be realized, a detailed understanding of the molecular mechanisms regulating the fundamental properties of ES cells, i.e. pluripotency, proliferation and differentiation, is required. In the present paper, we review the evidence that PI3K (phosphoinositide 3-kinase)-dependent signalling plays a role in regulation of both ES cell pluripotency and proliferation.