Appropriate biogenesis and subcellular trafficking of tRNA are essential for decoding the genome and for regulating the proteome in response to nutrient availability and stress. Hundreds of gene products function in tRNA biology and mutations in numerous of these gene products cause a variety of human disorders ranging from metabolic diseases, to neuromuscular diseases, and to cancer. Yet, there is a very incomplete understanding of tRNA biology. To address this information gap, we conducted a screen of nearly the entire essential and unessential yeast proteome and identified 162 novel proteins that function in tRNA biology (Wu et al., 2015). Although we identified gene products that function in virtually every step of tRNA biosynthesis and subcellular dynamics, we are particular interested in those proteins that function in tRNA nuclear export because it is essential that tRNAs be efficiently transported to the cytoplasm where protein synthesis occurs, but mutants missing the known tRNA nuclear exporters, los1Δ, msn5Δ, and los1Δ msn5Δ are perfectly viable. Surprisingly, we identified Mex67 (vertebrate TAP), Mtr2 (vertebrate p15), and Crm1 (vertebrate Exportin-1) as potential tRNA nuclear exporters. Previous studies in yeast and vertebrate cells have shown that Mex67 and Mtr2 form heterodimers that function in mRNA nuclear export, whereas Crm1 functions in nuclear export of proteins possessing a leucine-rich NE motif. Three lines of evidence support the role of these proteins in tRNA nuclear export: (1) as determined by Northern analysis, at the nonpermissive temperature, mex67-5, mtr2-x, and crm1-1 ts mutants each accumulate unspliced tRNAs and (2) as determined by FISH, each mutant has enhanced tRNA nuclear pools; (3) furthermore, los1Δ and crm1-1 have synthetic growth defects and Mex67 can completely substitute for Los1 when it is over-expressed in los1Δ cells. Thus, it appears that there are at least 3 parallel pathways that function to export tRNAs from their site of synthesis in the nucleus to their site of function in the cytoplasm, underscoring genetic redundancy for this essential cellular process. Preliminary data indicate that Mex67/Mtr2 and Crm1 are more error prone in tRNA nuclear export than Los1, raising questions regarding tRNA quality control during the nuclear export process. We are currently conducting in vivo biochemical analyses to determine whether tRNAs can be cross-linked to Mex67/Mtr2 and/or Crm1 as they exit the nucleus.