Adults are biologically responsive to context, and their responses to particular situations may differ across cultures. However, are preschoolers’ biological systems also responsive to situational contexts and cultures? Here, we show that children’s neurobiological stress responses, as indexed by salivary cortisol, are activated and responsive to psychosocial stressors relevant to their sociocultural emphases. By examining cortisol changes across different contexts among 138 preschoolers living in the United States, China, and Japan, we found that an achievement-related stressor elicited an increased cortisol response among Chinese preschoolers, whereas interpersonal-related stressors elicited an increased cortisol response among Japanese preschoolers. By contrast, U.S. preschoolers showed decreased cortisol responses after these stressors but consistently higher levels of anticipatory responses to separation at the beginning of each session. Our findings suggest that children’s neurobiological stress systems may be a critical biological mechanism allowing societal-level cultural phenomena to be embodied in individual-level responses, even among preschoolers.