Synthetic di-and tri-palmitoylated bacterial lipopeptide analogs (BLpA) can enhance HLA-I-restricted immune responses. Here we show that BLpA indirectly promote antigen-driven differentiation of naive CD4 + T lymphocytes in vitro, with mechanisms that require DC and are inhibited by CTLA-4/Ig. In mixed cultures of cord blood-derived PBMC and allogeneic DC, P 3 CSK 4 lipopeptide facilitated the transition from CCR7 + / CD45RA + /CD62L + to CCR7 -/CD45RA -/CD62L dim T cells with kinetics significantly exceeding those obtained with the unlipidated CSK 4 analog. Moreover, P 3 CSK 4 and P 2 CSK 4 , but neither the mono-palmitoylated PCSK 4 analog nor the CSK 4 peptide, increased the frequency of IFN-c-producing T cells expanded under similar conditions. Along with this, P 2 CSK 4 and P 3 CSK 4 , but not PCSK 4 , restored the in vitro antigenicity of MDP-OspA, a non-immunogenic analog of Borrelia burgdorferi major outer surface lipoprotein A, and enhanced the frequency of in vitro expanded T cells specific for the tetanus toxoid (TT) and hepatitis B surface antigen (HBsAg) peptides TT 947-967 and HBsAg 19-33 and for TT. Altogether, BLpA bearing at least two ester-bonded palmitoyl side chains indirectly enhance antigen-driven CD4 + T cell differentiation. BLpA adjuvanticity is independent of covalent bonding to Ag and Ag formulation. This information may be helpful to generate more potent recombinant vaccines.