Immunostaining is one of the advantageous methods for the qualitative analysis of cellular markers of interest. Immuno-stained cells are typically analyzed by fluorescence microscopy or flow cytometry. Flow cytometry has the advantage of being able to process large numbers of cells in a short time thus enhancing its quantitative capacity. The staining protocol typically includes fixation of cells followed by permeabilization, blocking procedures to reduce non-specific binding of the label, and staining with specific antibodies labeled directly or indirectly with fluorescence-tags. Important controls include staining with a relevant non-immune antibody to identify any non-specific imminent globally binding and measurements in the absence of any fluorescent tag to detect non-specific sources of fluorescence. The most common source of non-specific fluorescence is caused by autofluorescence of naturally occurring chemicals within the cells of interest. In this study, we found high levels of cellular autofluorescence in mouse embryonic fibroblasts, at levels that interfered with the detection of a number of cellular antigens using common fluorophores. This autofluorescence was detected in three of the four fluorescence channels restricting useful analysis to only one channel (red) on the instrument. The study highlights an important limitation to immunostaining techniques and reinforces the need for the use of a thorough set of controls to ensure specificity of quantitative analysis.