The process of tRNA splicing entails removal of an intron by TSEN (tRNA-splicing endonuclease) and ligation of the resulting exon halves to generate functional tRNAs. In mammalian cells, the RNA kinase CLP1 (cleavage and polyadenylation factor I subunit) associates with TSEN and phosphorylates the 3' exon at the 5' end in vitro, suggesting a role for CLP1 in tRNA splicing. Interestingly, recent data suggest that the ATP-binding and/or hydrolysis capacity of CLP1 is required to enhance pre-tRNA cleavage. In vivo, the lack of CLP1 kinase activity leads to progressive motor neuron loss and accumulation of novel 5' leader-5' exon tRNA fragments. We have extended the investigation of the biochemical requirements in pre-tRNA splicing and found that β-γ-hydrolysable ATP is crucial for the productive generation of exon halves. In addition, we provide evidence that phosphorylation of the TSEN complex components supports efficient pre-tRNA cleavage. Taken together, our data improve the mechanistic understanding of mammalian pre-tRNA processing and its regulation.