Abstract: Cooperation is wide-spread and has been postulated to drive major transitions in evolution. A cooperator pays a cost to benefit others, and when reciprocated, it gains a net benefit. However, Darwinian selection favors "cheaters" that consume benefits without paying a fair cost. Many cooperative systems have evolved sophisticated cheater recognition/exclusion mechanisms. How did cheater-resisting mechanisms evolve from simple cooperative systems? To address this question, I created a genetically tractable cooperative system that can be observed as it evolves, step-by-step, from its inception toward increased stability. It consists of two engineered non-mating yeast strains - a red-fluorescent R strain that requires adenine and releases lysine and a yellow-fluorescent Y strain that requires lysine and releases adenine. I observed that: the system is viable - able to grow from low-density to saturation in the absence of adenine and lysine supplements, over a wide range of conditions; system viability requirements could be calculated from growth, death, and metabolic properties of the two cooperating strains; the system evolved increased system viability: the minimum cell density required for system viability was reduced 100-fold. My group will: discover the diversity of changes that increase system viability. Pro-cooperation changes must act through benefiting self and/or partner. Properties of evolved strains will be measured and their relative contributions to enhanced cooperation will be quantified. determine mechanisms of cheater tolerance. After introducing a cheater that consumes but does not release metabolites, we will select for cooperator/cheater cocultures with increased cheater tolerance and delineate mechanisms. investigate the possibility of spatial structure stabilizing cooperation. We will compare viability requirements and cheater tolerance of the cooperative system in a well-mixed liquid culture (no spatial structure) with those on an agar pad (with spatial structure). We hope to quantitatively understand the evolution of cooperation and cheater tolerance. Public Health Relevance: Cooperation is a fundamental biological phenomenon. For instance, the human body relies on cooperation between different cell types. Diseases can be caused either by cheaters (such as cancer cells) that destroy normal cooperation or by formation of undesired cooperation (such as those found among infecting viruses or bacteria). Discovering and quantifying the importance of mechanisms that drive the evolution of cooperation and cheating may reveal strategies to stabilize or destabilize cooperation.