Although amino acids can function as signaling molecules in the regulation of many cellular processes, mechanisms surrounding L-threonine involvement in embryonic stem cell (ESC) functions have not been explored. Thus, we investigated the effect of L-threonine on regulation of mouse (m)ESC self-renewal and related signaling pathways. In L-threonine-depleted mESC culture media mRNA of self-renewal marker genes, [(3)H]thymidine incorporation, expression of c-Myc, Oct4, and cyclins protein was attenuated. In addition, resupplying L-threonine (500 μM) after depletion restores/maintains the mESC proliferation. Disruption of the lipid raft/caveolae microdomain through treatment with methyl-β-cyclodextrin or transfection with caveolin-1 specific small interfering RNA blocked L-threonine-induced proliferation of mESCs. Addition of L-threonine induced phosphorylation of Akt, ERK, p38, JNK/SAPK, and mTOR in a time-dependent manner. This activity was blocked by LY 294002 (PI3K inhibitor), wortmannin (PI3K inhibitor), or an Akt inhibitor. L-threonine-induced activation of mTOR, p70S6K, and 4E-BP1 as well as cyclins and Oct4 were blocked by PD 98059 (ERK inhibitor), SB 203580 (p38 inhibitor) or SP 600125 (JNK inhibitor). Furthermore, L-threonine induced phosphorylation of raptor and rictor binding to mTOR was completely inhibited by 24 h treatment with rapamycin (mTOR inhibitor); however, a 10 min treatment with rapamycin only partially inhibited rictor phosphorylation. L-threonine induced translocation of rictor from the membrane to the cytosol/nuclear, which blocked by pretreatment with rapamycin. In addition, rapamycin blocked L-threonine-induced increases in mRNA expressions of trophoectoderm and mesoderm marker genes and mESC proliferation. In conclusion, L-threonine stimulated ESC G(1)/S transition through lipid raft/caveolae-dependent PI3K/Akt, MAPKs, mTOR, p70S6K, and 4E-BP1 signaling pathways.