AbstractSummary: This study reports establishment of an in vitro culture system for E5.5 mouse embryos that supports development to the gastrulation stage and allows the use of experimental approaches to study gastrulation during mouse embryogenesis. Recent experiments suggest that the extraembryonic tissues may play a critical role for gastrulation from as early as E5.5. To apply whole embryo culture to E5.5 embryos and analyze gastrulation, it is essential to optimize the conditions so that most of the embryos develop to the gastrulation stage in culture. For this purpose, we established a protocol in which embryos were isolated using micromanipulator and cultured with 50–75% rat serum. Although cultured embryos tended to grow a larger extraembryonic portion, more than 80% of them developed the primitive streak and induce mesoderm, which corresponds to the mid‐streak stage. genesis 37:38–43, 2003. © 2003 Wiley‐Liss, Inc.