Excised plant tissues (explants) can regenerate new shoot apical meristems in vitro, but regeneration rates can be inexplicably variable. Light affects rates of shoot regeneration, but the underlying mechanisms are poorly understood. Here, excised Arabidopsis cotyledons were dark–light shifted to define the timing of explant light sensitivity. Mutants and pharmacological agents were employed to uncover underlying physiological and genetic mechanisms. Unexpectedly, explants were most light sensitive during the initial hours post-excision with respect to shoot regeneration. Only ∼100 µmol m−2 s−1 of fluorescent light was sufficient to induce reactive oxygen species (ROS) accumulation in new explants. By 48 h post-excision, induction of ROS, or quenching of ROS by xanthophylls, increased or decreased shoot regeneration, respectively. Phytochrome A-mediated signalling suppressed light inhibition of regeneration. Early exposure to blue/UV-A wavelengths inhibited regeneration, involving photoreceptor CRY1. Downstream transcription factor HY5 mediated explant photoprotection, perhaps by promoting anthocyanin accumulation, a pigment also induced by cytokinin. Surprisingly, early light inhibition of shoot regeneration was dependent on polar auxin transport. Early exposure to ethylene stimulated dark-treated explants to regenerate, but inhibited light-treated explants. We propose that variability in long-term shoot regeneration may arise within the initial hours post-excision, from inadvertent, variable exposure of explants to light, modulated by hormones.