Cooperation between unrelated individuals is a key element of human behavior. So how can we explain the great variation in cooperative norms that has been observed across cultures? A potential answer comes from understanding the cognitive underpinnings of cooperation: individuals internalize the cooperative or selfish strategies that are successful in their daily social interactions. Thus, when forces such as repetition, reputation and sanctions effectively promote prosociality, they also cause individuals to adopt cooperation as a default response; and this intuitive predisposition towards cooperation then continues to operate even in one-shot anonymous interactions beyond the reach of these mechanisms. Here we provide experimental evidence for this hypothesis by creating in vitro cultures of cooperation or defection in the laboratory. Our subjects play a series of repeated Prisoner’s Dilemma games constructed to make cooperation either an advantageous or disadvantageous strategy. In doing so, we immerse our subjects in environments where both themselves and those around them are consistently cooperating or defecting. We then test whether these behaviors become internalized by examining behavior in a subsequent battery of one-shot anonymous interactions. As predicted, subjects randomized into the cooperative environment are substantially more prosocial afterward, as well as more likely to punish selfishness. We provide evidence that this acculturation effect is driven by the remodeling of cooperative intuitions. We also show that the baseline behavior of American college students (who developed under strong institutions) matches that of our in vitro cooperative environment. Furthermore, this effect extends beyond economic games: our in vitro cooperative culture causes subjects to be more trusting, as measured by a standard survey instrument used in cross-cultural studies. These results provide direct evidence for the social heuristics hypothesis and shed light on the co-evolution of norms and institutions.