Multicellular 3-dimensional (3D) in vitro models of normal breast tissue to use in cancer initiation studies are lacking. We present a model incorporating 3 of the major functional cell types of breast, detail the phenotype and document our breast cancer initiation studies. Myoepithelial cells and fibroblasts were isolated and immortalised from breast reduction mammoplasty samples. Tri-cultures containing the non-tumorigenic luminal epithelial cells HB2, in parallel with HB2 overexpressing variants of HER family proteins, together with myoepithelial cells and fibroblasts were established in collagen 1. The effect of silencing ERβ1 in Myo1089 cells and the guanine nucleotide exchange factor DOCK4 in fibroblasts were also examined in these cultures. Phenotype was compared to normal breast tissue. Characterisation showed polarised epithelial structures with lumen formation and basement membrane production, similar to normal breast tissue. Similar to normal breast tissue, characterisation showed polarised epithelial structures with lumen formation and basement membrane production. The importance of including myoepithelial cells and fibroblasts in maintaining these structures was demonstrated. Overexpression of HER2 and HER2/3 resulted in a change in dramatic change in phenotype and development of DCIS-like structures. We established this model was amenable to genetic engineering by overexpressing HER2 and HER3 in HB2 cells and knocking out ERβ1 in Myo1089 cells and DOCK4 in fibroblasts using siRNA/shRNA techniques, respectively, although silencing these molecules did not affect phenotype. In summary, we have established an in vitro model of normal human breast tissue that includes three of the major functional breast cell types cultured in a physiologically relevant 3D matrix. Validating morphology and protein expression profiles against ‘gold standard’ human breast tissue confirmed this is a robust in vitro model of normal breast tissue offering an alternative and cost-effective method of studying genes and processes involved in breast cancer initiation.