CD20 is expressed in approximately one- half of pediatric acute lymphoblastic leukemia (ALL) cases with B-cell precursor (BCP) origin. We observed that it is occasionally up-regulated during treatment. To understand the impact of this on the potential effectiveness of anti-CD20 immunotherapy, we studied 237 CD10(+) pediatric BCP-ALL patients with Berlin-Frankfurt-Munster (BFM)-type therapy. We analyzed CD20 expression changes from diagnosis to end-induction, focusing on sample pairs with more than or equal to 0.1% residual leukemic blasts, and assessed complement-induced cytotoxicity by CD20-targeting with rituximab in vitro. CD20-positivity significantly increased from 45% in initial samples to 81% at end-induction (day 15, 71%). The levels of expression also increased; 52% of cases at end-induction had at least 90% CD20(pos) leukemic cells, as opposed to 5% at diagnosis (day 15, 20%). CD20 up-regulation was frequent in high-risk patients, patients with high minimal residual disease at end-induction, and patients who suffered later from relapse, but not in TEL/AML1 cases. Notably, up-regulation occurred in viable cells sustaining chemotherapy. In vitro, CD20 up-regulation significantly enhanced rituximab cytotoxicity and could be elicited on prednisolone incubation. In conclusion, CD20 up-regulation is frequently induced in BCP-ALL during induction, and this translates into an acquired state of higher sensitivity to rituximab. This study was registered at http://www.clinicaltrials.gov as #NCT00430118.