Flow cytometry is a technique for analyzing individual cells in solution based on light scatter and the expression of cellular markers. After being introduced into the flow cytometer, the cells are directed in single file past an interrogation point, where one or more lasers are focused. As each cell passes through the interrogation point, it scatters the laser light, and at the same time any fluorophore-labeled antibodies that are bound to the cell emit fluorescence. The resultant signals are then directed by a series of mirrors and filters towards a detector array, yielding data with which to characterize the cell population. A similar technique to flow cytometry, known as fluorescent-activated cell sorting (FACS), both identifies and isolates distinct cellular sub-populations, allowing for their use in downstream assays. The types of samples analyzed by flow cytometry include cell cultures, tissue homogenates, and blood, all of which must take the form of a single-cell suspension. While the sample preparation method will vary depending on the starting material, general good practice recommendations include passing the cells through a strainer and using resuspension buffers that contain DNase and EDTA and are free of Ca2+/Mg2+ ions. These steps serve to prevent the formation of clumps that could otherwise block the flow cytometer or generate misleading results. Once the cells are in suspension, researchers can proceed to immunostaining. If the aim of the flow cytometry experiment is to detect only cell surface markers, such as the cluster of differentiation (CD) antigens used for characterizing immune cells, the samples are typically blocked and then incubated with antibody reagents. However, if intracellular targets are of interest, fixation and permeabilization are necessary (prior to blocking) to ensure antibodies can access their targets. When working with immune cells, the inclusion of an Fc receptor (FcR) blocking step will help to minimize background signal by preventing antibodies from binding to FcRs via their Fc regions. A typical flow cytometry experiment will detect 5–10 different markers meaning that researchers must take care when it comes to antibody selection. While direct detection with fluorophore-labeled primary antibodies can both shorten the immunostaining protocol and allow for several antibodies sharing the same host species to be used in combination, indirect detection can offer greater flexibility for panel design. A broad range of secondary antibodies for flow cytometry is now available, including isotype- or subclass-specific antibodies, and antibodies labeled with tandem dyes. Fluorophore selection is equally as important as antibody selection. Because the signal from any fluorophore used in a multicolor flow cytometry panel will be measured across all of the instrument’s detectors, it is important to choose fluorophores that are spectrally distinct. In addition, controls are essential to address sources of off-target fluorescence. These should include unstained controls for evaluating autofluorescence, fluorescence-minus-one (FMO) controls to determine spread, and compensation controls to remove spillover. Isotype controls (antibodies that share the same host and isotype as analyte-specific antibodies but do not recognize the target of interest) are also useful for investigating whether antibodies are binding non-specifically to off-target components.