This unit describes a protocol for the differentiation of human embryonic stem cells (hESCs). To generate spin embryoid bodies (EBs), known numbers of hESCs are deposited into low‐attachment, round‐bottomed 96‐well plates in a serum‐free medium supplemented with growth factors. The cells are then aggregated by centrifugation, initiating formation of EBs of uniform size. The spin EBs generated using this technique differentiate efficiently and synchronously along the lineages preferentially induced by the combinations of growth factors to which the cells are exposed. The 96‐well format permits an assessment of the effects of different combinations of growth factors in the same experiment, facilitating the optimization of differentiation conditions for any given cell type. Up to 40 plates can be set up within a couple of hours by one experimenter, and aliquots of the differentiating EBs can be harvested at intervals and subjected to analyses using a variety of techniques. Curr. Protoc. Stem Cell Biol. 4:1D.3.1‐1D.3.23. © 2008 by John Wiley & Sons, Inc.