ABSTRACT T-cell functional avidity is a crucial determinant for efficient pathogen clearance. Although recombinant DNA priming coupled with a vaccinia-vectored vaccine (VACV) boost has been widely used to mount robust CD8 ⁺ T-cell responses, how VACV boost shapes the properties of memory CD8 ⁺ T cells remains poorly defined. Here, we characterize the memory CD8 ⁺ T cells boosted by VACV and demonstrate that the intrinsic expression of MyD88 is critical for their high functional avidity. Independent of selection of clones with high-affinity T-cell receptor (TCR) or of enhanced proximal TCR signaling, the VACV boost significantly increased T-cell functional avidity through a decrease in the activation threshold. VACV-induced inflammatory milieu is not sufficient for this improvement, as simultaneous administration of the DNA vaccine and mock VACV had no effects on the functional avidity of memory CD8 ⁺ T cells. Furthermore, reciprocal adoptive transfer models revealed that the intrinsic MyD88 pathway is required for instructing the functional avidity of CD8 ⁺ T cells boosted by VACV. Taking these results together, the intrinsic MyD88 pathway is required for the high functional avidity of VACV-boosted CD8 ⁺ T cells independent of TCR selection or the VACV infection-induced MyD88-mediated inflammatory milieu. IMPORTANCE Functional avidity is one of the crucial determinants of T-cell functionality. Interestingly, although it has been demonstrated that a DNA prime-VACV boost regimen elicits high levels of T-cell functional avidity, how VACV changes the low avidity of CD8 ⁺ T cells primed by DNA into higher ones in vivo is less defined. Here, we proved that the enhancement of CD8 ⁺ T cell avidity induced by VACV boost is mediated by the intrinsic MyD88 pathway but not the MyD88-mediated inflammatory milieu, which might provide prompts in vaccine design.