The impact of shipping temperatures and preservation media used during transport of either peripheral blood mononuclear cells (PBMCs) or Jurkat cells was assessed, in view of implementing of a proficiency testing scheme on mononuclear cell viability. Samples were analyzed before and after shipment at different temperatures (ambient temperature, dry ice, and liquid nitrogen) and in different preservation media (serum with cryoprotectant, commercial cryopreservation solution, and room temperature transport medium). Sample quality was assessed by viability assays (Trypan Blue dye exclusion, flow cytometry, Cell Analysis System cell counting (CASY)), and by ELISpot functional assay. The liquid nitrogen storage and shipment were found to be the most stable conditions to preserve cell viability and functionality. However, we show that alternative high quality shipment conditions for viable cells are dry ice shipment and commercial cryopreservation solution. These were also cost-efficient shipment conditions, satisfying the requirements of a proficiency testing scheme for viable mononuclear cells. Room temperature transport medium dramatically and adversely affected the integrity of mononuclear cells.