tRNAs are the central adaptor molecule in translation and require a wide variety of post-transcriptional modifications to fulfill their functions. The model gram-negative Escherichia coli K-12 is one of the handfuls of bacteria where all the tRNA modifications and corresponding genes have been characterized. This work dissects epistatic relationships between tRNA modification genes in E. coli by conducting a synthetic lethal screen revealing 5 pairs of modifications that cannot be deleted in combination when cells are grown in rich media, and 15 pairs of modifications that lead to growth defects when deleted in combination. The truA gene involved in the insertion of Psi residues at positions 38 to 40 in multiple tRNAs gave the highest number of synthetic lethality phenotypes that could be complemented by the expression of the truA gene in trans and in some cases suppressed by the overexpression of target tRNAs. A pilot phenotype screen of strains lacking two tRNA modifications genes viable in rich media lead to the identification of growth conditions that exhibited poor growth. This work lays the foundation to dissect the role of modifications in tRNA quality control.