Although the antigen expression patterns of childhood acute lymphoblastic leukemia (ALL) are well known, little attention has been given to standardizing the diagnostic and classification criteria. We retrospectively analyzed the flow cytometric data from a large study of antigen expression in 1,774 children with newly diagnosed ALL in JPLSG. T- and B-lineage ALL accounted for 13 and 87% of childhood ALL cases, respectively. Cytoplasmic CD3 and CD7 antigens were positive in all T-ALL cases. More than 80% of T-ALL cases expressed CD2, CD5 and TdT. In B-lineage ALL, the frequencies of early pre-B, pre-B, transitional pre-B and B-ALL were 81, 15.5, 0.6 and 2.9%, respectively. More than 90% of early pre-B ALL cases expressed CD19, CD79a, CD22, CD10 and TdT. CD34 was expressed in three-fourths of early pre-B ALL cases. The frequencies of TdT and CD34 expression were lower in pre-B ALL than in early pre-B ALL. B-ALL showed less frequent expression of CD22, CD10, CD34 and TdT than other B-lineage ALL cases. Expression of CD13 and CD33, aberrant myeloid antigens, was significantly more frequently associated with B-lineage ALL than with T-ALL. Based on this retrospective study of antigen expression in 1,774 de novo childhood ALL cases in JPLSG, we propose standardized clinical guidelines for the immunophenotypic criteria for diagnosis and classification of pediatric ALL.