The prognosis of infant B-cell acute lymphoblastic leukemia (iB-ALL) remains dismal, especially in patients harboring the MLL-AF4 (KTM2A-AFF1) rearrangement, which arises prenatally in early hematopoietic stem/progenitor cells (HSPCs) and accounts for 80% of iB-ALL and 10% of non-infant cases. MLL-AF4+ B-ALL shows a bimodal localization of the MLL gene breakpoint within the MLL break cluster region, and two subgroups of patients based on the gene expression pattern of the HOXA/MEIS cluster have been identified. The pathogenic mechanisms in MLL- AF4+ B-ALL are challenging to study functionally due to the absence of faithful human cellular models recapitulating the disease phenotype and latency. Here, we assess the molecular contribution and leukemogenic capacity of MLL breakpoints occurring in either intron 10 (MLL i10 , centromeric) or intron 12 (MLL i12 , telomeric) in ontogenically-different human HSPCs sourced prenatally (fetal liver) and neonatally (cord blood). CRISPR-Cas9-induced MLL-AF4 (MA) targeting either MLL i10 (M i10 A) or MLL i12 (M i12 A) causes MA-driven in vitro myeloid immortalization in both fetal liver- and cord blood-CD34+ HSPCs. The centromeric location of the MLL breakpoint, but not the cellular ontogeny, determined the expression of HOXA/MEIS1 genes in MLL-edited cells. Centromeric MLL breakpoints endowed enhanced myeloid clonogenic replating to MLL- edited CD34+ HSPCs. The cellular ontogeny and the location of the MLL breakpoint also influenced the capacity of MLL-edited CD34+ HSPCs to initiate pro-B-ALL in vivo, which faithfully recapitulated the molecular, transcriptomic and methylome profiles of patients with primary MA+ iB-ALL. Our data provide key insights into the cellular and molecular leukemogenic determinants of MA+ iB-ALL. This dataset contains processed RNAseq and DNA methylation data from the abovementioned study.