Household pesticide exposure during pregnancy has been associated with a more than 2-fold increased risk in infant leukemia, and chlorpyrifos (CPF) is among the most frequently applied insecticides. During early fetal development, liver is a hematopoietic organ with majority of cells being CD34⁺ hematopoietic stem cells (CD34⁺HSC). The in utero injury to CD34⁺HSC has been known to underlie the pathogenesis of several blood disorders, often involving rearrangements of the mixed-lineage leukemia (MLL) gene on 11q23. In this study, we evaluated the leukemogenic potential of CPF in human fetal liver-derived CD34⁺HSC. Specifically, exposure to 10 μM CPF led to decrease in viability, inhibition in proliferation and induction of DNA double-strand breaks (DSBs) and occurrence of MLL⁺ rearrangements. In particular, we observed CPF-mediated cell cycle disturbance as shown by G0/G1 arrest, in contrast to etoposide (VP-16), an anticancer drug used as a positive control and known to induce G2/M arrest. Further study on mechanisms underlying DNA DSBs and MLL⁺ rearrangements revealed that CPF might act as topoisomerase II poison, a mechanism of action similar to VP-16. On the other hand, CPF was also shown to induce early apoptosis through active caspase-3 activation, a pathway known to underlie DNA DSBs and MLL⁺ translocations. Our data indicate that in utero injury of CD34⁺HSC by CPF may contribute to the increased risk of infant leukemia. Future work will elucidate the mechanism and the type of CPF-induced MLL⁺ translocations in HSC.