Summary Flow cytometry (FCM) testing in hematopoietic malignancies is essential for diagnosing the disease, assessing treatment efficacy, and determining tumor cell involvement. FCM testing with a cerebrospinal fluid (CSF) is often requested for the assessment of central nervous system (CNS) infiltration in acute lymphoblastic leukemia/lymphoblastic lymphoma (ALL/LBL) and diffuse large B-cell lymphoma (DLBCL). However, cell numbers of the specimen are often limited; preparation of the testing material requires careful pre-processing, unlike cell-rich specimens such as bone marrow. The following points should generally be considered for small cell volume specimens. ① Determination of tumor cell amount in the specimen: use an automated hematology analyzer or manually count and calculate the number of cells using a calculation board before the FCM analysis. ② Specimen preservation for morphology inspection: consider appropriate methods for morphology inspection among the wedge method, the compression and stretching method, the spinner method, the collecting cell method, and the spontaneous sedimentation method, depending on the type and volume of specimens. ③ Optimization of washing and concentration process: excessive washing should be avoided as much as possible to prevent cell loss during washing. Cell concentration should also be adjusted according to the required test volume. ④ Using the minimal number of antibodies necessary: reduce the number of tubes to increase the number of cells per tube for practical testing. ⑤ Determination of the cell gate by considering cell morphology: consider the ratio and structural features of the tumor cells. In addition to the points described above, a case analysis of CSF specimens is presented in this manuscript.