Background: Signaling studies in cell lines are hampered by non-physiological alterations obtained in vitro. Physiologic primary tumor cells from patients with leukemia require passaging through immune-compromised mice for amplification. The aim was to enable molecular work in patients' ALL cells by establishing siRNA transfection into cells amplified in mice. Results: We established delivering siRNA into these cells without affecting cell viability. Knockdown of single or multiple genes reduced constitutive or induced protein expression accompanied by marked signaling alterations. Conclusion: Our novel technique allows using patient-derived tumor cells instead of cell lines for signaling studies in leukemia.