The stromal microenvironment is central to chronic lymphocytic leukemia (CLL) pathogenesis. How leukemic cells condition the stroma to enhance its chemoattractant properties remains elusive. Here we show that mouse and human CLL cells promote the contact-independent stromal expression of homing chemokines. This function was strongly enhanced in leukemic cells from Em-TCL1 mice lacking the pro-oxidant p66Shc adaptor, which develop an aggressive disease with organ infiltration. We identified Interleukin (IL) -9 as the soluble factor, negatively modulated by p66Shc, responsible for the chemokine-elevating activity of leukemic cells on stromal cells. IL-9 blockade in Em-TCL1/p66Shc-/- mice resulted in a decrease in the nodal expression of homing chemokines, which correlated with decreased leukemic cell invasiveness. IL-9 levels were found to inversely correlate with residual p66Shc in the p66Shc-deficient human CLL cells (n=52 patients). p66Shc reconstitution in CLL cells normalized IL-9 expression and neutralized their chemokine-elevating activity. Notably, high IL-9 expression in CLL cells directly correlates with lymphadenopathy, liver infiltration, disease severity and overall survival, emerging as an independent predictor of disease outcome. Our results demonstrate that IL-9 modulates the chemokine landscape in the stroma, and that p66Shc, by regulating IL-9 expression, tunes the ability of leukemic cells to shape the microenvironment, thereby contributing to CLL pathogenesis.Copyright © 2020 American Society of Hematology.