Improper compensation leads to incorrect gate frequency calculations. Cells were stained with a series of reagents, including CD123 conjugated to phycoerythrin, but no reagent on the fluorescein isothiocyanate (FITC) channel. (Left) The distribution of CD123 vs FITC is shown. Essentially, no events show up in the FITC channel. (Right) The compensation setting between phycoerythrin and FITC was reduced by 0.4% to illustrate the impact of undercompensation. Such an error in the estimation of the proper setting could easily be seen by using a compensation control that was not as bright as the CD123^bright cells. Tell-tale signs of undercompensation are apparent, including the curved upward appearance of the distribution and the highly correlated distribution (narrow diagonal) of the brightest events. The same gate now shows more than 1% “positive” events, despite there being no FITC fluorescence on these cells.