Engineered cellular therapy products are among the more complex of immunotherapeutic modalities because they are living drugs. As such, flow cytometry-based assays have become essential tools to demonstrate biological critical quality attributes such as identity, cell health and fitness, and potency. One of the biggest challenges in designing and executing flow cytometry-based assays is identifying and controlling variability. A poorly controlled and highly variable assay can increase invalid and re-test rates, or worse, cause a manufacturing process to appear out of control or a drug product to appear unstable. Identifying and mitigating sources of variability begins during initial assay design, as part of QbD for method development, and should continue to be a focus through life cycle management. Here, we will discuss expected and unexpected sources of variability and control strategies in flow cytometry-based assays through representative case studies.