Regulation of embryonic stem (ES) cells by amino acids has not been well characterised, yet knowledge of amino acid metabolism and transport in stem cell differentiation will be critical in the optimisation of differentiation protocols for ES cells and improving the production of ES cell-derived cells for regenerative medicine. The ability of L-proline to induce the differentiation of mouse ES cells into a second pluripotent cell population, early primitive ectoderm-like (EPL) cells, suggest active roles for amino acids in early embryo development and pluripotent cell regulation. Radioactive amino acid uptake assays, gene expression analysis and immuno-fluorescent techniques identified a system A amino acid transporter, SNAT2 (SLC38a2) as the major transporter required for l-proline transport into mouse ES cells. Inhibiting the transport of amino acids by SNAT2 blocked the ability of l-proline to induce the formation of primitive ectoderm-like cells from ES cells, suggesting the requirements of L-proline uptake into ES cells through SNAT2 in this differentiation process. SNAT2 has been shown to be a ‘transceptor’, capable of regulating intracellular pathway activity in response to nutrient availability, and, in conjunction with other transporters at the cell surface, forms an interface with the extracellular environment that controls not only the intracellular pool of amino acids in ES cells but potentially establishments of cell function and cell identity. The expression of SNAT2 and another system A transporter, SNAT1 in mouse ES cells and within the mouse embryo has shown dynamic patterns of expression and novel subcellular localisations. These data suggests previously unidentified mechanisms by which amino acid transporters can regulate cell and embryo functions. Understanding the repertoire of amino acid transporters present in pluripotent cells and in the embryo is a first step to unravelling the roles of amino acids play in ES cell maintenance, differentiation and in embryogenesis.