When grafted in vivo a population of fetal thymic epithelial cells, marked by the monoclonal antibodies MTS20 and MTS24, can generate all major thymic epithelial cell subtypes. Furthermore, the resultant thymic organoid can recruit T cell precursors and support their differentiation into mature CD4⁺ and CD8⁺ T cells. The work presented here evaluates the potential of MTS20⁺ thymic epithelial progenitor cells to form the basis of a thymus equivalent in vitro. To assess the ability of the MTS20⁺ thymic epithelial progenitor cell (TEPC) population to support T cell differentiation in vitro, improvements were made to the established reaggregate fetal thymic organ culture (RFTOC) method that permitted the reliable generation and culture of TEPC-based RFTOC (TEPOC). Subsequent analysis demonstrated that both MTS20⁺ and MTS20⁺ fetal thymic epithelial cells were able to support the differentiation of αβ and γδ T cells and also supported the differentiation of several other haematopoietic populations including dendritic cells and thymic macrophages. However, while immunofluorescence analysis showed that MTS20⁺ cells were able to differentiate in vitro to generate all major thymic epithelial cell populations, only limited differentiation was observed in MTS20⁺ cell-based cultures. Strikingly, the TEPOCs were able to form organised epithelial structures in vitro, characterised by clearly distinguished adjacent medullary and cortical areas. No evidence of organisation was seen in MTS20⁺ cell-based cultures. Together, these data establish that MTS20⁺ but not MTS20⁺ thymic epithelial cells can generate functional in vitro thymus-equivalents that recapitulate the epithelial and haematopoietic landscape of the wild-type thymus. Furthermore, the unique organisational ability inherent within the TEPOCs may be useful as an in vitro model of the processes governing thymus organisation.