Lymphokines interleukin-4 (IL4) and IL13 exert overlapping biological activities via the shared use of the IL4 receptor alpha-chain and signal transducer and activator of transcription 6 (Stat6). Stat6 is critical for T-helper 2 cell differentiation, B-cell Ig class switch, and allergic diseases; thus, understanding its regulation is of central importance. Phosphorylation is crucial for Stat activity. Whereas Stat6 is phosphorylated on Tyr(641), less is known about serine or threonine. We demonstrate in primary human T-cells (>95% CD3+) that IL4 and for the first time IL13 induce Stat6 serine but not threonine phosphorylation that closely paralleled early IL4 receptor alpha-chain activation (10 min). Stat6 uniquely fails to share a positionally conserved Stat serine phosphorylation sequence; however, known phosphoacceptor sites are proline-flanked. Alanine substitutions of these conserved residues revealed that the transactivation domain, which localized Ser(756) but not Ser(827) or Ser(176), is the IL4-regulated site based on phosphoamino acid analysis. Tyr(641) was dispensable for IL4-mediated serine phosphorylation, suggesting that dimerization is not preconditional. Only Stat6 Y641F variant showed a significant effect on IL4-inducible Cepsilon DNA-binding and reporter gene expression. Lastly, recent work has shown that protein phosphatase 2A negatively regulates Stat6. We propose this target residue(s) is distinct from Ser(756) and may be proximal to Tyr(641) at Thr(645), a residue conserved only among Stat6 members. The phosphomimic variants T645E or T645D ablated Stat6 activation, whereas polar uncharged substitutions (Gln or Asn) and additional mutants (Ala, Val, or Phe) showed no effect. These findings suggest that Stat6 has mechanisms of regulation distinct from other Stats.