Sézary Syndrome (SS) is a rare and aggressive epidermotropic cutaneous T-cell lymphoma (CTCL) defined by erythroderma, pruritis and a circulating atypical CD4+ T-cell clonal population. The diversity of Sézary cells (SC) phenotype and genotype possibly reflects either plasticity or heterogeneity that is difficult to assess, as SC are difficult to expand with very few cell lines available. Therefore, we developed six new defined culture conditions allowing the amplification of SC defined by their immunophenotype and monoclonality in 4 of 7 patients. The SC expansion in response to different culture conditions by addition of several cytokines and stromal cells is heterogeneous between patients. This shows the importance of tumor microenvironment for tumor SC growth. Engraftment of SC into immunodeficient NOD.Cg-Prkdc(scid)Il2rg(tm1Wjll)/SzJ (NSG) mice was achieved in 2 of 14 cases. Secondary xenograft by subcutaneous injection mimicked several clinical features of SS with dermal infiltration, epidermotropism and blood spreading. Such models permitted to assess the intra-individual heterogeneity of patient SC. Such subclones sharing the same TCR gene rearrangement evolved independently according to culture condition and/or after xenografting. This clonal selection was associated with phenotypic differences and limited genomic evolution both in vitro and in vivo. The long-term amplification of SC allowed the development of eight new SC lines derived from four different patients. They represent the cell of origin diversity of SC cells and new tools to evaluate their functional properties. Indeed, SC lines demonstrate differential responses to therapies (romidepsin, doxorubicin and vorinostat) according to the cells of origin of SC. The new in vivo model we developed mimicking both skin and blood involvement of SS represents a new preclinical model to test therapeutic agents as well as the mechanisms regulating the balance between blood and skin compartments of SC cells.