MEDII, a conditioned medium derived from the human hepatocarcinoma cell line, HEPG2, has previously been shown to produce a 95% homogenous population of neuroectoderm cells from mouse embryonic stem (mES) cells1. Importantly, this directed differentiation proceeds via a series of embryologically relevant cell populations including early primitive ectoderm-like (EPL) cells, definitive ectoderm, neuroectoderm and finally, terminally differentiated neurons2. MEDII however is a complex medium composed of various unknown constituents. Fractionation experiments have identified L-proline and the IL-6 cytokine Leukaemia Inhibitory Factor (LIF) as bioactive molecules within MEDII involved in this directed differentiation3. Therefore an L-proline-based protocol in combination with mouse LIF and nodal inhibitor (SB431542) was used to drive the differentiation of mESCs towards neuroectoderm in order to study molecular mechanisms associated with various aspects of early neurogenesis. mES cells were cultured as embryoid bodies for 9 days in medium containing specific and time-dependent combinations of L-proline, LIF and SB431542. Differentiation of the mES cells was assessed by marker analysis using qPCR, flow cytometry and immunofluorescence to quantify the production of EPL cells, definitive ectoderm and neuroectoderm. The results from this work suggest that this protocol induces the sequential expression of embryologically relevant cell populations. Furthermore, time-lapse imaging and phase-contrast microscopy showed that approximately 80% of these embryoid bodies produced neural phenotypes. These are the first findings that suggest that L-proline acts like a growth factor to induce the differentiation of mESCs to neuroectoderm via EPL cell and definitive ectoderm intermediates.