Chronic Lymphocytic Leukemia (CLL) cells disseminate into supportive tissue microenvironments. To investigate the mechanisms involved in leukemic cell tissue retention we developed a 3D bone marrow (BM) microenvironment that recreates CLL - BM-stromal cells interactions inside a scaffold within a bioreactor. Our system allows the parallel analysis of CLL cells retained inside the scaffold and those released in the presence/absence of pharmacological agents, mimicking tissue and circulating cell compartments, respectively. CLL cells can be retained within the scaffold only in the presence of microenvironmental elements, which through direct contact down-regulate the expression of HS1 cytoskeletal protein in CLL cells. Consist with this, the expression of HS1 was lower in CLL cells obtained from patients' BM versus CLL cells circulating in the PB. Moreover, we demonstrate that CLL cells with inactive-HS1, impaired cytoskeletal activity and a more aggressive phenotype are more likely retained within the scaffold despite the presence of Ibrutinib, whose mobilizing effect is mainly exerted on those with active-HS1, ensuing dynamic cytoskeletal activity. This differential effect would not otherwise be assessable in a traditional 2D system and may underlie a distinctive resistance of single CLL clones. Notably, CLL cells mobilized in the peripheral blood of patients during Ibrutinib therapy exhibited activated HS1, underscoring that our model reliably mirrors the in vivo situation. The 3D model described herein is suitable to reproduce and identify critical CLL-BM interactions, opening the way to pathophysiological studies and the evaluation of novel targeted therapies in an individualized manner.