Using this PLA approach, we were able to confirm or deny the existence of the endogenous ETV6-RUNX1 fusion protein in pre-B lymphoblastic cell lines and, more interestingly, in pre-B lymphoblasts from leukemic patients. Additionally, we were able to demonstrate the molecular proximity of CBFB, a well-known cofactor of RUNX1, with ETV6-RUNX1. The optimized PLA procedure can be readily applied to other non-adherent hematological cells, from cell lines or patients’ cells to detect fusion proteins as well as protein interactions.