Hanging drop (HD) cultures were carried out with a drop volume of either 20 or 30 mu l. An incubation period of 3 days was determined to be appropriate for the formation of well-controlled embryoid bodies (EBs), and the initial cell number was identified as the most critical factor in the growth and neuronal cell differentiation of EBs. (c) 2012, The Society for Biotechnology, Japan. All rights reserved.