Stem cells are an important in vivo model for embryo development. Our lab and others have shown that several amino acids can function as signalling molecules which regulate stem-cell differentiation. L-Proline (L-Pro) together with the cytokine leukaemia inhibitory factor (LIF) induces mouse embryonic stem cell (mESCs) proliferation and formation of a distinct pluripotent cell population ^1 2 3 - early primitive ectoderm-like (EPL) cells which have the characteristics of embryonic primitive ectoderm. On the other hand, self-renewal of mESCs depends on another amino acid, L-Threonine (L-Thr), which controls the G1-to-S phase transition via Akt and MAPK signalling pathways; these pathways are important during early embryo development. Our aim is to determine whether mESC self-renewal and rate of differentiation is dependent on the concentration of specific amino acids. mESCs were cultured in medium containing LIF ± different concentrations of L-Proline or L-Threonine. Cell type was identified by marker analysis using qPCR. Our results showed that higher concentrations of L-Pro prolonged the expression of markers of primitive ectoderm and accelerated Primitive Streak formation compared to lower concentrations. On the other hand, increased concentration of L-Thr sustained self-renewal of mESCs even when the concentration of LIF was reduced from 1000 U/mL to 330 U/mL. Our results indicate that amino acids can act like growth factors in combination with the cytokine LIF to affect the self-renewal and differentiation properties of mESCs in a concentration-dependent manner. These data provide evidence for the role of these amino acids in cell differentiation during early embryo development.