Starting confluence can be a critical factor in the efficient generation of definitive endoderm. This is especially important when the differentiation procedure starts with the plating of a single cell suspension of undifferentiated pluripotent stem cells. Because confluence can be a relatively subjective measurement, we prefer to plate a specific number of cells into each well when starting a differentiation run. The optimal number of cells is 2.1 x 105 cells/cm². This translates to 800,000 cells in a 12-well plate well, or 2 million cells in each well of a 6-well plate. When this number of cells is plated in mTeSR1 with 10 µM Y-27632, the user can expect to see a monolayer of cells at greater than 80% confluence after overnight culture. There is more tolerance on the higher side of this cell number than on the lower side. Plating up to 1.2 million cells in a well of a 12-well plate will not adversely affect differentiation efficiency. Reducing the number of cells to 600,000 cells per well may result in reduced performance. Because confluence is a critical factor to achieving highly efficient definitive endoderm differentiation, plating single cells as a monolayer is preferred over plating of clumps or colonies. The size distribution of clumps or colonies tends to be significant with traditional scraping techniques. The size of these clumps can potentially influence the differentiation potential of the resulting colonies thus introducing variability into the system. Furthermore, it is difficult to achieve a target starting confluence when plating clumps, a problem that is largely avoided by working with single cells.