Depletion of gut microbiome bacteria after antibiotic treatment promotes pB-ALL development in predisposed mice in the absence of natural infectious stimuli. (A) Untreated Pax5^+/– mice housed in SPF facilities did not develop leukemia. Treatment of Pax5^+/– mice housed in SPF facilities with antibiotics resulted in pB-ALL development in 47.82% of mice. **P = .0055 using a Fisher’s exact test. (B) pB-ALL–specific survival curves of Pax5^+/– mice treated with antibiotics and exposed to common infections (green line; n = 27), Pax5^+/– mice treated with antibiotics and housed in SPF facility conditions (blue line; n = 23), and Pax5^+/– mice not treated with antibiotics and housed in SPF facility conditions (orange line; n = 12) are shown. Log-rank (Mantel-Cox) test (P = .2629) was used to evaluate survival curves between untreated Pax5^+/– mice and Pax5^+/– mice treated with antibiotics. (C) Representative fluorescence-activated cell sorting plots of B-cell (CD19⁺B220⁺ and B220⁺IgM^+/–) subsets in PB and BM from diseased Pax5^+/– mice treated with antibiotics (exposed [CF] and not exposed [SPF facility] to common infections) compared with an age-matched control WT mouse are shown. (D) The Venn diagram illustrates recurrently mutated genes in Pax5^+/– leukemic cells identified by whole-exome sequencing. Mutated genes in leukemias arising in Pax5^+/– mice treated with antibiotics and housed either in CFs (green circle) or in SPF facilities (blue circle) are compared with leukemias derived from untreated Pax5^+/– mice kept in an infectious environment (red circle). ns, not significant.