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Flow cytometry (FCM) is used for quantification of minimal residual disease (MRD) in acute lymphoblastic leukemia (ALL) through discriminating leukemic B-lymphoblasts from normal B-cell precursor counterparts “hematogones.” Neuropilin-1 (NRP-1)/CD304 is a vascular endothelial growth factor receptor implicated in the progression of hematological malignancies. We evaluated NRP-1/CD304 as MRD and prognostic marker in pediatric precursor B-ALL using FCM. Seventy children with precursor B-ALL and 40 control children were enrolled. CD304 percentage and fluorescence intensity were significantly higher in precursor B-ALL at diagnosis compared with controls. In total, 28 of 70 (40%) precursor B-ALL patients at diagnosis were CD304+ (group A), whereas 42/70 (60%) patients were CD304− (group B). Group A showed higher incidence of lymphadenopathy and TEL-AML1 fusion gene than group B. CD304 was reevaluated in group A patients at day 28 postinduction chemotherapy which revealed 12/28 (42.9%) patients with persistent CD304+ expression (MRD+; group A1) and 16/28 (57.1%) patients who turned CD304− (MRD−; group A2). At diagnosis, group A1 showed lower incidence of TEL-AML1 fusion gene and higher risk stratification than group A2. NRP-1/CD304 expression by FCM is efficient in discriminating leukemic B-lymphoblasts from hematogones, a stable leukemia-associated phenotype for MRD monitoring, and a putative poor prognostic marker in pediatric precursor B-ALL.