Umbilical cord blood is a key source of stem cells for transplantation and regenerative medicine. To maximize each cord blood sample, it is important to analyze its cellular populations. Many cord blood banks focus on counts of total nucleated cells and/or cells that carry the CD34 antigen, but this limited focus does not give a true estimation of cord blood content, quality and appropriateness for use. This protocol is the first of its kind to enable a comprehensive investigation of cord blood cellular populations. Using multicolor flow cytometry it is possible to examine expression of 26 antigens--including hematopoietic markers CD45 and CD34, immune markers CD19 and CD3, and HLA--using a total of only 1 x 10(6) cells from each unit for both pre- and post-processing. The samples are stained, lysed, washed and analyzed flow cytometrically. This method also provides valuable information beyond cord blood composition and quality; for example, it can also be used to assess whether maternal factors affect CD34(+) cell numbers. Collection, processing, cryopreservation and initial flow cytometry take approximately 5 h.