Minimal residual disease (MRD) constitutes the most important prognostic factor in B-cell precursor acute lymphoblastic leukemia (BCP-ALL). Flow cytometry is widely used in MRD assessment, yet little is known regarding the effect of different immunophenotypic subsets on outcome. In this study of 200 BCP-ALL patients, we found that a CD34-positive, CD38 dim-positive, nTdT dim-positive immunophenotype on the leukemic blasts was associated with poor induction therapy response and predicted an MRD level at the end of induction therapy (EOI) of ≥ 0.001. CD34 expression was strongly and positively associated with EOI MRD, whereas CD34-negative patients had a low relapse risk. Further, CD34 expression increased from diagnosis to relapse. CD34 is a stemness-associated cell-surface molecule, possibly involved in cell adhesion/migration or survival. Accordingly, genes associated with stemness were overrepresented among the most upregulated genes in CD34-positive leukemias, and protein–protein interaction networks showed an overrepresentation of genes associated with cell migration, cell adhesion, and negative regulation of apoptosis. The present work is the first to demonstrate a CD34-negative immunophenotype as a good prognostic factor in ALL, whereas high CD34 expression is associated with poor therapy response and an altered gene expression profile reminiscent of migrating cancer stem-like cells.