Aim Flow cytometric crossmatch (FCXM) method requires larger number of cells and serum volume compared to CDC method. FCXM is widely used for living donor transplantation. However, it is not commonly used for preliminary crossmatches for deceased donor transplant candidates, partly due to limited number of donor cells. We developed an effective FCXM method using about 10% of cell number and serum volume that is used in most of the flow laboratories, i.e., 300,000–500,000 cells and 30–100μL of sera. Methods We compared 3 different kinds of washing fluids for cell recovery after washing. After 5 times of washing, cell recovery was much higher for PBS–2% FBS (94%) and PBS–0.2% BSA (80%) compared to PBS (11%), and PBS–0.2% BSA was selected as washing fluid. Total lymphocytes with >90% purity, which were isolated from peripheral blood using Ficoll separation and EasySep negative selection (STEMCELL Technologies) were used for the test. Test was set up using 50,000 cells (in 5.0–7.5μL volume) and 5μL sera in 96 well U type microplate (3 color T/B stain in single well), and 20% of the final volume (20/100μL; theoretically, 10,000/50,000 cells) was acquired for analysis using FACSCalibur flow cytometer and automated microtiter plate HTS module (Becton Dickinson). Cell recovery was tested by 4 different technologists and actual cell number available for flow cytometric analysis in routine testing of 20 deceased donor crossmatches by 2 technologists was analyzed. Results Cell recovery in FCXMs tested by 4 different technologists was 77% in average and was not different among technologists (78.5%, 78.4%, 76.9%, 74.5%). Actual cell number analyzed in routine testing of 20 deceased donor crossmatches by 2 technologists was higher in negative control serum (AB type serum) than in positive control serum (high-PRA pooled serum): for total/T/B lymphocytes, 8453/6801/1439 cells in negative and 6537/5713/631 cells in positive control serum. The number of cells acquired was adequate for analysis in most of the cases, and if needed, larger number of cells can be acquired by increasing the volume aspirated through automated HTS module. Conclusion Flow crossmatches using very small number of cells (50,000 cells) and serum volume (5μL) in microplate can be used effectively for deceased donor as well as for living donor transplantation.