Progenitor B cells deficient in Pax5 are developmentally multipotent, suggesting that Pax5 is necessary to maintain commitment to the B-cell lineage. Commitment may be mediated, in part, by Pax5 repression of myeloid-specific genes. To determine whether Pax5 expression in multipotential cells is sufficient to restrict development to the B-cell lineage in vivo, we enforced expression of Pax5 in hematopoietic stem cells using a retroviral vector. Peripheral blood analysis of all animals reconstituted with Pax5-expressing cells indicated that more than 90% of Pax5-expressing cells were B220+ mature B cells that were not malignant. Further analysis showed that Pax5 completely blocked T-lineage development in the thymus but did not inhibit myelopoiesis or natural killer (NK) cell development in bone marrow. These results implicate Pax5 as a critical regulator of B-versus T-cell developmental fate and suggest that Pax5 may promote commitment to the B-cell lineage by mechanisms that are independent of myeloid gene repression. © 2003 by The American Society of Hematology.