During the early stages of mammalian development, cells of the embryo transition through the inner cell mass, primitive-ectoderm, definitive-ectoderm and neurectoderm before giving rise to cells that make up the nervous system. Due in part to the complexity of differentiation, the small size of the embryo and its relative inaccessibility in utero, the molecular mechanisms underlying the formation of these cell populations, and thus development of the neural lineage, are relatively poorly understood. We developed a protocol using mESCs to recapitulate mammalian nervous system development in vitro using L-proline. D3 and 46C-Sox1-GFP mESC cell lines were cultured as embryoid bodies (EBs) with time-dependent additions of 400 μM L-proline and 10 μM SB431542 over 9 days. On Day 9, EBs were seeded in serum-free conditions and allowed to differentiate for a further 6 days, after which they were assessed for neural cells (D3: 61±14% and 46C: 68±9% (n≥3)). Using qPCR (data expressed as peak log2 fold change, n≥3), mESCs differentiated to Dnmt3b+ (3±0.5) and Fgf5+ (5±0.3) primitive-ectoderm between Days 3–5, followed by Penk1+ (1±0.6) and Pard6b+ (2±0.1) definitive-ectoderm between Days 5–7. From Days 5–9, a Sox1+ population of neurectoderm (2.5±0.3) was produced, at the expense of Mixl1+ mesendoderm (-2.1±1.2). By Day 9, flow cytometry analysis showed that 68±2% cells cultured in L-proline were Sox1-GFP+, and immunofluorescence imaging indicated that this expression was maintained in some cells at the core of EBs cultured until Day 15. Taken together, neurectoderm production from mESCs was achieved prior to the addition of SB431542, thus providing evidence for an instructive model of neurectoderm induction in the presence of L-proline. This is the first study, to our knowledge, that shows that a simple amino acid acts like a typical growth factor to induce differentiation at multiple stages of mammalian development.