Human embryonic stem cells (hESCs) replicate in vitro by the process of self-renewal, whilst maintaining their pluripotency. Understanding the pathways involved in the regulation of this process will assist in developing fully-defined conditions for the robust proliferation of hESCs necessary for therapeutic applications. We previously demonstrated that sphingosine-1-phosphate (S1P) plays an important role in survival and proliferation of hESCs. and here the key signaling pathways and downstream targets of S1P were investigated in a representative cell line (Shef 4). A significant rise in ERK1/2 activation with S1P treatment was witnessed in hESCs maintained on murine embryonic fibroblasts (MEFs) exhibiting significantly higher levels of active ERK1/2 than those grown on Matrigel. RT-PCR and microarray analysis of micro-dissected, non-differentiated hESC revealed 1049 differentially expressed genes in S1P treated preparations compared with controls (n = 3). S1P regulated apoptosis through several BCL-2 family members, including BAX and BID, with increased expression of cell cycle progression genes associated with proliferation of hESC cultures. S1P treatment also increased expression of cell adhesion genes specifically cadherins and integrins. However, gene expression associated with pluripotency was decreased with S1P treatment indicating that an increased rate of hESC turnover (higher proliferation and lower apoptosis) may be balanced by an increased susceptibility to differentiate.