Pluripotent embryonic stem (ES) cells, a potential source of somatic precursors for cell therapies, cause tumors after transplantation. Studies of mammalian carcinogenesis using nuclear magnetic resonance (NMR) spectroscopy have revealed changes in the choline region, particularly increased phosphocholine (PCho) content. High PCho levels in murine ES (mES) cells have recently been attributed to cell pluripotency. The phosphoinositide 3-kinase (PI3K)/Akt pathway has been implicated in tumor-like properties of mES cells. This study aimed to examine a potential link between the metabolic profile associated with choline metabolism of pluripotent mES cells and PI3K/Akt signaling. We used mES (ES-D3) and murine embryonal carcinoma cells (EC-F9) and compared the metabolic profiles of 1) pluripotent mES (ESD0), 2) differentiated mES (ESD14), and 3) pluripotent F9 cells. Involvement of the PI3K/Akt pathway was assessed using LY294002, a selective PI3K inhibitor. Metabolic profiles were characterized in the extracted polar fraction by (1)H NMR spectroscopy. Similarities were found between the levels of choline phospholipid metabolites (PCho/total choline and PCho/glycerophosphocholine [GPCho]) in ESD0 and F9 cell spectra and a greater-than five-fold decrease of the PCho/GPCho ratio associated with mES cell differentiation. LY294002 caused no significant change in relative PCho levels but led to a greater-than two-fold increase in PCho/GPCho ratios. These results suggest that the PCho/GPCho ratio is a metabolic trait shared by pluripotent and malignant cells and that PI3K does not underlie its development. It is likely that the signature identified here in a mouse model may be relevant for safe therapeutic applications of human ES cells.