Flow cytometry (FC) is of increasing importance for the analysis of cerebrospinal fluid (CSF) lymphocytes because of its ability to detect a large spectrum of cellular characteristics (granularity, volume, surface antigen expression) even in small amounts of cells. Data on CSF FC in children are very limited. Here, we summarize our 3-year experience of CSF FC routinely performed in pediatric patients with assumed inflammatory central nervous system (CNS) disease. Among 109 samples sent for analysis, flow cytometric detection of major leukocyte subsets was possible in 78% (85 out of 109), which exceeds the 31% rate of our retrospective microscopic pediatric control group. Apart from physiologic lymphocytes (100%) or monocytes (48%), 11 out of these 85 samples showed granulocytes, two showed proliferated monocytes, and nine displayed proliferated lymphocytes. In most children, the proliferated lymphocytes consisted of a polyclonal population of CD4+ and CD8+ T cells. Compared with literature data, eight children showed abnormally composed lymphocyte subsets (surface antigen expression) within the main lymphocyte population. However, none of these changes was specific for distinct diseases or allowed a distinction between patients with and without primary inflammatory processes. These data suggest that CSF FC may be the most effective modality to differentiate major CSF leukocyte subsets. At present, further differentiation of distinct cell populations, such as proliferated lymphocytes, is of limited clinical impact. This may, however, gain increasing interest in the future.