Abstract <p>Tissue spheroids, which are three-dimensional cellular aggregates, represent a powerful tool in biomedical research and tissue engineering. This review summarizes contemporary methods of spheroid biofabrication, including the hanging drop technique, use of nonadhesive surfaces, microfluidic systems, bioreactors, magnetic and acoustic levitation, chemical inducers of aggregation, and hydrogel matrices. Their advantages, limitations, and applications are discussed in detail. Special attention is paid to the process of spheroid fusion as a key step in the creation of larger tissue constructs as well as to methods for the quantitative assessment of its kinetics. The core of the review is an analysis of the molecular mechanisms underlying spheroid formation and fusion. The key roles of cell adhesion molecules—such as E-cadherin, N-cadherin, and integrins—cytoskeletal elements (actin microfilaments and microtubules), signaling pathways (Wnt/β-catenin), and extracellular matrix components (collagen, fibronectin, laminin) are described. Current understanding of cell self-organization processes and the prospects for using spheroids to create functional tissue models and constructs in regenerative medicine and oncology research are discussed.</p>