Tubulin nucleation is a highly frequent event in microtubule (MT) dynamics but is poorly understood. In this work, we characterized the structural changes during the initial nucleation phase of dynamic tubulin. Using size-exclusion chromatography-eluted tubulin dimers in an assembly buffer solution free of glycerol and tubulin aggregates enabled us to start from a well-defined initial thermodynamic ensemble of isolated dynamic tubulin dimers and short oligomers. Following a temperature increase, time-resolved X-ray scattering and cryo-transmission electron microscopy during the initial nucleation phase revealed an isodesmic assembly mechanism of one-dimensional (1D) tubulin oligomers (where dimers were added and/or removed one at a time), leading to sufficiently stable two-dimensional (2D) dynamic nanostructures, required for MT assembly. A substantial amount of tubulin octamers accumulated before two-dimensional lattices appeared. Under subcritical assembly conditions, we observed a slower isodesmic assembly mechanism, but the concentration of 1D oligomers was insufficient to form the multistranded 2D nucleus required for MT formation.