Chemically cross-linked hydrogels constitute a novel injectable tissue engineering material. At present, one of the key problems is to find an appropriate initiator. This study evaluated the cytotoxicity in vitro of a water-soluble redox initiating system consisting of ammonium persulfate (APS) and N, N, N′, N′-tetramethylethylenediamine (TEM- ED). Gelation time of PEG diacrylate macromer in phosphate buffer saline solution was first adjusted to guarantee that the examined initiator concentrations are sufficiently high to trigger polymerization of macromers. NIH/3T3 fibroblasts were employed to examine cytotoxicity via MTT measurements and optical microscopic observations. It has been found that the combined APS/TEMED system exhibits negative cooperative effect, for the underlying cytotoxicity is even lower than that of APS or TEMED at certain concentrations.