AML1/Runx1 is a frequent target of human leukemia–associated gene aberration and encodes a transcription factor with nonredundant biologic functions in initial development of definitive hematopoiesis, T-cell development, and steady-state platelet production. AML1/Runx1 and 2 closely related family genes, AML2/Runx3 and AML3/Runx2/Cbfa1, present in mammals, comprise the Runt-domain transcription factor family. Although they have similar structural and biochemical properties, gene-targeting experiments have identified distinct biologic roles. To directly determine the presence of functional overlap among runt-related transcription factor (Runx) family molecules, we replaced the C-terminal portion of acute myeloid leukemia 1 (AML1) with that derived from its family members, which are variable in contrast to conserved Runt domain, using the gene knock-in method. We found that C-terminal portions of either AML2 or AML3 could functionally replace that of AML1 for myeloid development in culture and within the entire mouse. However, while AML2 substituted for AML1 could effectively rescue lymphoid lineages, AML3 could not, resulting in a smaller thymus and lymphoid deficiency in peripheral blood. Substitution by the C-terminal portion of AML3 also led to high infantile mortality and growth retardation, suggesting that AML1 has as yet unidentified effects on these phenotypes. Thus, the C-terminal portions of Runx family members have both similar and distinct biologic functions.