Cells of the inner cell mass (ICM) of the mouse blastocyst differentiate into 
the pluripotent epiblast or the primitive endoderm (PrE), marked by the 
transcription factors NANOG and GATA6, respectively. To investigate the 
mechanistic regulation of this process, we applied an unbiased, quantitative, 
single-cell-resolution image analysis pipeline to analyze embryos lacking or 
exhibiting reduced levels of GATA6. We find that Gata6 mutants exhibit a 
complete absence of PrE and demonstrate that GATA6 levels regulate the timing 
and speed of lineage commitment within the ICM. Furthermore, we show that GATA6 
is necessary for PrE specification by FGF signaling and propose a model where 
interactions between NANOG, GATA6, and the FGF/ERK pathway determine ICM cell 
fate. This study provides a framework for quantitative analyses of mammalian 
embryos and establishes GATA6 as a nodal point in the gene regulatory network 
driving ICM lineage specification.