tRNAs, nexus molecules between mRNAs and proteins, have a central role in translation. Recent discoveries have revealed unprecedented complexity of tRNA biosynthesis, modification patterns, regulation and function. In this Review, we present emerging concepts regarding how tRNA abundance is dynamically regulated and how tRNAs (and their nucleolytic fragments) are centrally involved in stress signalling and adaptive translation, operating across a wide range of timescales. Mutations in tRNAs or in genes affecting tRNA biogenesis are also linked to complex human diseases with surprising heterogeneity in tissue vulnerability, and we highlight cell-specific aspects that modulate the disease penetrance of tRNA-based pathologies.