The mouse blastocyst, at the time of implantation, has three distinct cell lineages: epiblast (EPI), trophoblast and primitive endoderm (PE). Interactions between these three lineages and their directional growth and migration are critical for establishing the initial asymmetries that result in anterior–posterior patterning of the embryo proper. We have re–investigated the timing of specification of the three lineages in relation to the differential allocation of progeny of the first two blastomeres to the embryonic versus abembryonic axis of the blastocyst. We find that the majority of cells of the inner cell mass (ICM) are specified to be EPI or PE by the mid 3.5 day blastocyst and that this is associated with localized expression of GATA–6 in the ICM. We propose a model for molecular specification of the blastocyst lineages in which a combination of cell division order, signal transduction differences between inner and outer cells and segregation of key transcription factors can produce a blastocyst in which all three lineages are normally set up in an ordered, lineage–dependent manner, but which can also reconstruct a blastocyst when division order or cell interactions are disturbed.