When treated with retinoic acid (RA), a human embryonal carcinoma (EC) cell line, NTera2 cl.D/1 (NT2), differentiates into several morphologically distinct cell types, which include terminally differentiated postmitotic central nervous system (CNS) neurons. Accumulating evidence has demonstrated the significant potential of NT2 cells in studies related to cancer therapy and neurodegenerative diseases. However, preparation of enriched NT2 neurons often requires a lengthy period (ca. five weeks) and depends largely on tedious techniques similar to those used for primary neuronal cultures. Here, we report a rapid protocol for the preparation of these human CNS neurons. Using the method of cell aggregation, enriched NT2 neurons can be obtained in approximately two weeks. We also demonstrated that cell aggregation reduced the time normally required for the induction of neuronal differentiation, as revealed by the early expression of neuronal markers. The period of RA treatment could also be reduced if NT2 cells were maintained as aggregates for a sufficient period of time. Taken together, our findings demonstrated that cell aggregation promoted RA-induced neuronal differentiation of NT2 cells and provided a rapid protocol for the efficient production of NT2 neurons. The ability to produce large quantities of human CNS neurons should facilitate future use of these neurons for basic research and applications in cell therapy.