Mutualisms (mutually beneficial symbioses) are ubiquitous in nature but have evolutional vulnerability because they are based on interactions that are not directly beneficial for their own fitness. A basis in proximate factor for explaining this paradox of ubiquity and vulnerability may exist but it remains to be elucidated. Insight can be gained by not only retracing the history of well-developed natural mutualisms, but also by observing the development of nascent synthetic mutualisms. As a simple example, we have recently simulated the establishment of a nascent mutualism using two genetically engineered auxotrophic strains of Escherichia coli. One strain, around 10 h after mixing with the partner strain, began to oversupply metabolites essential for the partner’s growth, eventually leading to continual growth of both strains. That is, the study demonstrated that an organism had the potential to adapt to the first encounter with another organism to establish a mutualism. Here in the poster presentation, we show that the synthetic mutualism did not decline but developed in 1,000 generations and discuss a specific proximate factor for it. We hope such strategy using synthetic symbiosis will become more widely used for exploring general bases in proximate factor.