What activity defines a star being on the main sequence?

Steady, controlled nuclear fusion of hydrogen into helium in the core.

The definitive characteristic of a star residing on the main sequence phase is the ongoing, stable, and controlled nuclear fusion process occurring deep within its core, where hydrogen nuclei are consistently transformed into helium nuclei. This sustained energy generation provides the outward pressure necessary to counteract the inward pull of gravity, establishing hydrostatic equilibrium. This stable burning phase represents the longest and primary part of a star's active life, accounting for about 90% of the total lifespan for Sun-like stars. Only when the core hydrogen supply is depleted does the star cease this defining activity and begin moving off the main sequence band on the Hertzsprung-Russell diagram.