Background: Breast cancer is a leading cancer in women worldwide. Many primary breast cancers are estrogen receptor positive (ER+) and responsive to anti-estrogenic therapies. These tumors can mutate estrogen receptors to survive, allowing the tumors to become more triple-negative-like and therefore more dangerous. Triple-negative breast cancer (TNBC) has been particularly challenging to treat due to its lack of estrogen (ER), progesterone (PR), and human epidermal growth factor (HER2) receptors. Due to the lack of treatment options, TNBC has a poor prognosis and contributes to a significant percentage of breast cancer mortalities. These ER mutants act more like TNBC, resulting in worse clinical outcomes. Current research on these ER mutants has been conducted using two-dimensional (2D), monolayer cell culture, which does not translate effectively in animal models, and ultimately, humans. Three-dimensional (3D) cell culture, which allows for the formation of spheroids, mimics actual tumors and provides results more consistent with tumor treatment in vitro. Due to the lack of research on these ER mutants in 3D culture, they must be characterized to determine baseline gene expression and behavior. After characterization, identifying changes resulting from drug treatment will be possible. Methods: Parental, ER+ MCF7-Luc cells, and daughter D538G and Y537S mutants were seeded at a density of 3000 cells/well in a low-attachment, round-bottomed 96-well plate. 48 hours and 7 days post-seeding, newly-formed spheroids were imaged. Using ImageJ analysis software, diameter, area, perimeter, circularity, aspect ratio, roundness, and solidity were determined. After 7 days in culture, spheres were collected for RNA extraction. Next, cDNA was synthesized, and qRT-PCR was performed to assess gene expression differences. Results: The wild-type ER+ spheres have smaller diameters, spherocity values closer to one, and are more compact. They express different levels of EMT markers from the controls, indicating alterations to signaling pathways. These 3D cultures vary in expression from the 2D cultures of the same cell lines. Conclusions: MCF-7 ER+ breast cancer cells aggregate more readily than ER+ mutants. The ER must be involved in signaling that promotes aggregation, as reduced ER signaling decreases the ability for spheroid formation. This phenomenon and the differences in gene expression may explain why mutants tend to behave in a more triple-negative manner. Cells cultured in 3D express some genes to different extents, confirming the importance of 3D culture for identifying future therapies – cells behave differently in different culturing contexts, 3D being more consistent with tumor behavior. Therefore, characterizing ER+ mutants prior to drug treatment studies is crucial to understanding how compounds affect cancer cells, as well as for identifying differences in various ER+ mutants for better treatment outcomes.