Conditional inactivation of Pbx1 in pro-B cells using CD19Cre does not prevent B-cell development. (A) Southern blot analysis of Pbx1 gene configurations in FACS-purified B cells (CD19⁺) in the bone marrow (left) and spleen (right) was performed on mice with the genotypes indicated above the gel lanes. The respective migrations of Pbx1 DNA fragments corresponding to the deleted (Δ), floxed (f), wild-type (+), and null (−) alleles are indicated to the right of the panel. The absence of floxed and wild-type alleles in CD19⁺ cells of Pbx1^f-−CD19Cre mice indicates complete Pbx1 deletion in B cells (lanes 3 and 8). (B) FACS analysis shows normal numbers of B220⁺IgM⁺ B cells and CD4⁺ and CD8⁺ single-positive T cells in the peripheral blood of Pbx1^f/−CD19Cre transgenic mice. (C) FACS analysis demonstrates that IgM^hiIgD^lo and IgM^loIgD^hi B-cell subsets are present at normal levels in the secondary lymphoid organs of Pbx1^f/−CD19Cre mice. (D) B-cell developmental subsets (Hardy fractions A-F) are normally represented in the bone marrow of Pbx1^f/−CD19Cre mice. (E) Role of Pbx1 during B-cell development. The B-cell developmental pathway originating from upstream HSCs and CLPs is shown schematically. Relative Pbx1 expression levels are illustrated below and depict the marked down-regulation of Pbx1 with progressive differentiation along the B-cell lineage. Genetic analyses in this report define a Pbx1-dependent stage of B-cell development, with a critical requirement for Pbx1 function at a point between HSCs and B-cell commitment.