The t(12;21) chromosomal translocation, targeting the gene encoding the RUNX1 transcription factor, is observed in 25% of pediatric acute lymphoblastic leukemia (ALL) and is an initiating event in the disease. To elucidate the mechanism by which RUNX1 disruption initiates leukemogenesis, we investigated its normal role in murine B-cell development. This study revealed 2 critical functions of Runx1: (1) to promote survival and development of progenitors specified to the B-cell lineage, a function that can be substituted by ectopic Bcl2 expression, and (2) to enable the developmental transition through the pre-B stage triggered by the pre-B-cell antigen receptor (pre-BCR). Gene expression analysis and genomewide Runx1 occupancy studies support the hypothesis that Runx1 reinforces the transcription factor network governing early B-cell survival and development and specifically regulates genes encoding members of the Lyn kinase subfamily (key integrators of interleukin-7 and pre-BCR signaling) and the stage-specific transcription factors SpiB and Aiolos (critical downstream effectors of pre-BCR signaling). Interrogation of expression databases of 257 ALL samples demonstrated the specific down-regulation of the SPIB and IKZF3 genes (the latter encoding AIOLOS) in t(12;21) ALL, providing novel insight into the mechanism by which the translocation blocks B-cell development and promotes leukemia.