B-cell acute lymphoblastic leukemia (B-ALL) is predominantly a childhood diseasewith approximately 75% of patients younger than 6 years of age. Currently, chemotherapy including VCR, DNR and Ara-C constitutes the major components of treatments, which is limited by its intolerable side effects. PI3Kδ, which is largely enriched in the hematopoietic system, is hyper-activated in most B-cell lymphoblastic leukemia and has attracted increasing interest as a target for therapy in B-cell leukemias. The first PI3Kδ specific inhibitor Idelalisib was just approved in US at July this year to treat patients with relapsed follicular B-cell non-Hodgkin lymphoma (FL) and relapsed small lymphocytic lymphoma (SLL), but the underlying antitumor mechanisms are not fully understood. Researchers in Jian Ding group in Shanghai Institute of MateriaMedica (CAS), cooperated with colleagues from Xcovery and Shanghai Children’s Medical Center, designed and synthesized novel PI3Kδ inhibitor X-370 with distinct binding model based on the crystal structure of p110δ. Here we report a novel PI3Kδ selective inhibitor X-370, which displays distinct binding mode with p110δ and blocks constitutively active or stimulus-induced PI3Kδ signaling. X-370 significantly inhibited survival of human B cell leukemia cells in vitro, with associated induction of G1 phase arrest and apoptosis. X-370 abrogated both Akt and Erk1/2 signaling via blockade of PDK1 binding to and/or phosphorylation of MEK1/2. Forced expression of a constitutively active MEK1 attenuated the antiproliferativeactivity of X-370. X-370 preferentially inhibited the survival of primary pediatric B-ALL cells displaying PI3Kδ-dependent Erk1/2 phosphorylation, while combined inhibition of PI3Kδ and MEK1/2 displayed enhanced activity. We conclude that PI3Kδ inhibition led to abrogation of both Akt and Erk1/2 signaling via a novel PI3K-PDK1/MEK1/2-Erk1/2 signaling cascade, which contributed to its efficacy against B-ALL. These findings support the rationale for clinical testing of PI3Kδ inhibitors in pediatric B-ALL and provide insights needed to optimize the therapeutic strategy.